GOT1 Human

What is GOT1 and what is its primary function in human metabolism?

GOT1 is a cytosolic enzyme that catalyzes the reversible transfer of an amino group from L-aspartate to α-ketoglutarate, forming oxaloacetate and L-glutamate. This enzyme plays essential roles in amino acid metabolism, cellular energy production via the malate-aspartate shuttle, glutamate metabolism, and maintenance of nitrogen balance. GOT1 is also known as cytosolic aspartate aminotransferase (cAST) and is expressed in various human tissues, with particularly high levels in the liver, heart, skeletal muscle, kidneys, and brain .

The enzyme participates in critical metabolic pathways that affect cellular bioenergetics, neurotransmitter metabolism, and response to cellular stress. Its activity is measurable in serum and often used as a biomarker for tissue damage, particularly in liver and cardiac conditions.

How does GOT1 differ from GOT2 in terms of cellular localization and function?

While GOT1 and GOT2 catalyze the same biochemical reaction, they differ significantly in their cellular localization and specific functions:

Both enzymes are critical for the malate-aspartate shuttle, which transfers reducing equivalents across the mitochondrial membrane, but they operate on opposite sides of this shuttle system. This complementary function allows for coordinated metabolic activities between cytosolic and mitochondrial compartments.

What are the normal reference ranges for GOT1 activity in human populations?

GOT1 activity in healthy human subjects typically ranges from 7-45 U/l . These reference ranges can vary depending on several factors:

• Analytical methodology

• Population demographics

• Age and sex

• Fasting status

• Pre-analytical variables

Research studies have found that the average GOT1 activity in healthy individuals is approximately 10 U/l, though this can vary across different populations . It's worth noting that GOT1 activity in rats (approximately 95 U/l) is almost 10-fold higher than in humans, which has important implications for translational research and extrapolating findings from rodent models to human applications .

These baseline differences must be considered when designing studies and interpreting results across species, particularly when developing therapeutic applications targeting GOT1 activity.

What significant genetic variants of GOT1 have been identified in human populations?

Genome-wide association studies have identified several genetic variants in the GOT1 gene, with the most notable being an in-frame deletion of three nucleic acids encoding asparagine at position 389 (c.1165_1167delAAC, resulting in p.Asn389del) . This deletion has distinctive characteristics:

• Primarily identified in the Amish population with a minor allele frequency (MAF) of 0.0052

• Absent in a sample of 647 outbred Caucasians, suggesting population specificity

• Conservation of the Asn389 residue among known mammalian cytosolic ASTs, indicating functional importance

• Significant reduction in enzymatic activity in carriers compared to non-carriers

The variant was discovered through a genome-wide association study of serum aspartate aminotransferase (AST) activity in 866 Amish participants, with subsequent genotyping in additional Amish samples (n=1932) identifying 20 more carriers .

Research suggests that carriers of this deletion have significantly lower AST activity levels (10.0±2.8 U/l) compared with homozygotes for the common allele (18.8±5.2 U/l), demonstrating the functional significance of this genetic variant .

How does the c.1165_1167delAAC deletion affect GOT1 protein expression and enzymatic activity?

The c.1165_1167delAAC deletion results in the loss of asparagine at position 389 (p.Asn389del) in the GOT1 protein, with profound effects on both protein expression and enzymatic function:

• Protein Expression: In vitro transient transfection studies comparing wild-type and mutant cAST showed that the mutant cAST protein was barely detectable in cells, suggesting that the deletion significantly affects protein stability or synthesis .

• Enzymatic Activity: Even after correction for the reduced expression levels, the mutant cAST demonstrated markedly diminished enzymatic activity compared to the wild-type protein .

• Mechanistic Impact: The asparagine residue at position 389 is highly conserved among mammalian cASTs, indicating its structural or functional importance. The deletion likely disrupts proper protein folding, stability, or catalytic function.

• Phenotypic Correlation: Despite the significant biochemical impact, research has not identified associations between this deletion and metabolic traits including serum fasting glucose, insulin, lipids, inflammatory markers, or sub-clinical markers of cardiovascular disease .

This suggests that while the variant significantly impacts the biochemical parameters of the enzyme, compensatory mechanisms may exist that prevent manifestation of overt metabolic phenotypes.

What are the appropriate experimental approaches for characterizing novel GOT1 variants?

Characterizing novel GOT1 variants requires a comprehensive approach combining genetic, biochemical, and functional analyses:

• Genetic Analysis:

  • Sequencing of GOT1 gene (exons, introns, and regulatory regions)

  • Genotyping using Custom TaqMan SNP Genotyping Assays or similar methods

  • Population frequency determination across diverse ethnic groups

• Expression Vector Construction:

  • PCR amplification of the full GOT1 protein-coding region with appropriate primers

  • Site-directed mutagenesis to introduce the variant of interest

  • Cloning into expression vectors (e.g., pFlag-CMV2 vector)

• In Vitro Protein Characterization:

  • Transient transfection into appropriate cell lines

  • Western blot analysis for protein expression levels

  • Enzyme activity assays with specific substrates

  • Protein stability and half-life determination

• Functional Studies:

  • Cell-based assays to assess impact on metabolic pathways

  • Assessment of impact on malate-aspartate shuttle function using techniques like the Peredox NADH biosensor

  • Measurement of glutamate handling capacity

For the previously characterized c.1165_1167delAAC variant, researchers used full-length human cAST cDNA as a template for PCR amplification with restriction enzyme-anchored primers, followed by the construction of wild-type and mutant expression vectors for transfection studies .

What is the role of GOT1 in ischemic stroke pathophysiology and potential therapeutic applications?

GOT1 has emerged as a potential therapeutic target for ischemic stroke based on its ability to reduce glutamate levels in blood and brain tissue . The pathophysiological rationale and therapeutic applications include:

• Glutamate Excitotoxicity Mechanism:

  • During ischemic stroke, excessive glutamate accumulates in the extracellular space

  • This leads to excitotoxicity and neuronal death

  • Blood glutamate scavenging is a protective strategy to reduce this excitotoxic effect

• GOT1-Mediated Mechanism:

  • GOT1 catalyzes the conversion of glutamate and oxaloacetate to α-ketoglutarate and aspartate

  • This reaction helps remove excess glutamate from the bloodstream

  • Lowering blood glutamate creates a concentration gradient that facilitates the efflux of glutamate from the brain to the blood

• Clinical Correlation:

  • Stroke patients with higher baseline GOT activity (average 17 U/l) showed reduced serum glutamate concentrations and significantly better neurological outcomes compared to patients with lower GOT activity (average 11 U/l)

• Therapeutic Approach:

  • Administration of recombinant GOT1 (rGOT1) alone or in combination with oxaloacetate

  • This approach has shown promise in animal models of ischemic stroke

  • Treatment efficacy was demonstrated even when delayed up to 2 hours after the onset of ischemia

The therapeutic potential of rGOT1 is particularly promising because it doesn't require blood-brain barrier penetration and doesn't act directly on neuronal glutamate receptors, avoiding the problems encountered with previous glutamate antagonists .

How does rGOT1 treatment affect serum and brain glutamate levels in experimental stroke models?

Studies in experimental stroke models have demonstrated that rGOT1 treatment significantly impacts both serum and brain glutamate levels through complementary mechanisms:

• Serum Glutamate Effects:

  • Administration of rGOT1 significantly increased systemic GOT activity 1 hour after administration

  • This elevated activity returned close to normal levels 24 hours after treatment

  • The increased GOT activity was associated with a reduction in serum glutamate concentrations within 6 hours

• Brain Glutamate Effects:

  • Quantitative analysis using Magnetic Resonance Spectroscopy (MRS) revealed that control animals had persistent increases in brain glutamate levels after middle cerebral artery occlusion

  • rGOT1 treatment significantly decreased glutamate levels in the brain parenchyma

  • This reduction in brain glutamate corresponded with reductions in infarct volume as assessed by MRI

• Temporal Profile:

  • Brain and serum glutamate levels appear to increase in the first 2 hours after reperfusion in experimental models

  • The therapeutic window for rGOT1 administration may extend up to 2 hours after the onset of ischemia

  • Protective effects were observed even when treatment was delayed until 1 hour after reperfusion

• Functional Outcomes:

  • The reduction in glutamate levels correlated with improved somatosensory function in treated animals

  • Protective effects persisted for at least 7 days after the ischemic event

These findings demonstrate that rGOT1 treatment creates a brain-to-blood glutamate efflux that protects neural tissue from excitotoxic damage.

What are the challenges in translating GOT1-based therapies from animal models to human clinical applications?

Several significant challenges exist in translating GOT1-based therapies from experimental models to human clinical applications:

Based on preclinical data, it is hypothesized that administering rGOT1 doses that increase systemic GOT activity by at least two or three-fold would be necessary for clinical efficacy in stroke patients .

How does GOT1 contribute to the malate-aspartate shuttle and cellular energy metabolism?

GOT1 plays a critical role in the malate-aspartate shuttle, a key mechanism for transferring reducing equivalents (NADH) from the cytosol into the mitochondria for ATP production:

• Shuttle Mechanism:

  • In the cytosol, GOT1 catalyzes the conversion of aspartate to oxaloacetate, which is then reduced to malate by malate dehydrogenase 1 (MDH1)

  • This reduction of oxaloacetate to malate is coupled with the oxidation of NADH to NAD+

  • Malate then enters the mitochondria via specific transporters

  • Inside the mitochondria, malate is converted back to oxaloacetate by MDH2, generating NADH

  • Mitochondrial GOT2 then converts oxaloacetate to aspartate, which can exit the mitochondria, completing the cycle

• Energetic Impact:

  • This shuttle effectively transfers reducing equivalents (NADH) from the cytosol to the mitochondria

  • Mitochondrial NADH feeds into the electron transport chain for ATP production

  • The shuttle is particularly important in tissues where direct NADH transport is limited

• Tissue-Specific Importance:

  • The malate-aspartate shuttle is especially active in highly aerobic tissues like heart, liver, and brain

  • Recent research has identified its importance in brown adipose tissue thermogenesis

  • GOT1 activity affects the cytosolic NAD+/NADH ratio, influencing multiple metabolic pathways

This shuttle system represents a sophisticated mechanism for coordinating cytosolic and mitochondrial metabolism in response to cellular energy demands, with GOT1 serving as a critical enzymatic component of this process.

What is the role of GOT1 in brown adipose tissue thermogenesis?

Recent research has revealed an important role for GOT1 in brown adipose tissue (BAT) thermogenesis through specific regulatory and metabolic mechanisms:

• Expression Pattern:

  • GOT1 is highly enriched in cold-activated brown and beige adipocytes in both mice and humans

  • Transcriptome analysis identified Got1 as specifically upregulated in response to cold exposure

• Regulatory Pathway:

  • Cold exposure activates a specific signaling cascade in brown adipocytes:

    • β-adrenergic receptor activation → cAMP increase → PKA activation → PGC-1α/NT-PGC-1α induction

    • PGC-1α/NT-PGC-1α bind to the GOT1 gene promoter at estrogen-related receptor (ERR) binding motifs

    • This results in increased GOT1 expression

• Functional Significance:

  • GOT1 activates the malate-aspartate shuttle in brown adipocytes

  • This shuttle appears to support thermogenesis by:

    • Facilitating continued cytosolic glycolysis by regenerating NAD+

    • Supporting mitochondrial oxidative metabolism

    • Potentially coordinating with UCP1-mediated thermogenesis

• Experimental Evidence:

  • Studies with a genetically encoded NADH biosensor (Peredox) demonstrated that GOT1 influences cytosolic NADH dynamics in brown adipocytes

  • GOT1 activity was shown to be inhibited by aminooxyacetate (AOA)

  • Overexpression of GOT1 in BAT enhanced thermogenic capacity

This research identifies GOT1 as a cold-inducible metabolic regulator in brown adipose tissue and suggests potential targets for metabolic research focused on energy expenditure and obesity treatments.

How can researchers effectively monitor intracellular NADH dynamics related to GOT1 activity?

Monitoring intracellular NADH dynamics related to GOT1 activity requires specialized techniques that enable real-time assessment of metabolic processes:

• Genetically Encoded Biosensors:

  • The Peredox biosensor system provides a powerful tool for monitoring cytosolic NADH dynamics

  • Peredox consists of a GFP variant linked to a bacterial NADH-binding protein (Rex)

  • Upon binding NADH, Rex undergoes a conformational change that increases green fluorescence

  • Ratiometric measurement is enabled by tandem fusion with mCherry

• Experimental Implementation:

  • The biosensor can be expressed in relevant cell types (e.g., brown adipocytes)

  • Real-time fluorescence microscopy allows monitoring of NADH levels in living cells

  • Interventions affecting GOT1 activity can be assessed by measuring changes in the fluorescence ratio

  • Specific inhibitors like aminooxyacetate (AOA) can confirm GOT1 involvement

• Analytical Approaches:

  • Sequential addition of substrates and inhibitors:

    • Glucose to establish baseline metabolism

    • AOA to inhibit GOT1/GOT2

    • Iodoacetate/pyruvate to inhibit glycolysis

  • Monitoring fluorescence changes in the same cell over time provides direct evidence of GOT1's impact on NADH metabolism

• Combined Methodologies:

  • Integration with metabolomics to correlate NADH changes with substrate/product levels

  • Genetic manipulation (overexpression/knockdown) of GOT1 to validate specificity

  • Comparison between different cell types or conditions to assess context-dependent effects

This approach enables researchers to directly visualize the impact of GOT1 activity on cellular NAD+/NADH dynamics, providing insights into its role in metabolic pathways and energy metabolism.

What are the most effective approaches for studying GOT1 function in human cell and tissue models?

Studying GOT1 function in human cell and tissue models requires a multifaceted approach combining genetic, biochemical, and functional methodologies:

• Cell Culture Models:

  • Primary human cells (hepatocytes, adipocytes, neurons)

  • Relevant cell lines (HepG2, neuronal lines, brown adipocyte lines)

  • Patient-derived cells (e.g., fibroblasts from individuals with GOT1 variants)

  • iPSC-derived specialized cell types for tissue-specific studies

• Genetic Manipulation Strategies:

  • CRISPR/Cas9 gene editing to:

    • Create knockout models

    • Introduce specific variants (e.g., c.1165_1167delAAC)

    • Add reporter tags to endogenous GOT1

  • RNA interference for transient knockdown

  • Overexpression systems using appropriate vectors

• Functional Readouts:

  • Enzymatic activity assays

  • Metabolomics profiling to assess glutamate/aspartate ratios and TCA cycle intermediates

  • Seahorse XF analysis for mitochondrial function and glycolytic flux

  • Live-cell imaging with metabolite sensors (e.g., Peredox for NADH)

• Tissue-Specific Approaches:

  • For brown adipose tissue: cold exposure models, β-adrenergic stimulation, thermogenic capacity measurement

  • For neural tissue: glutamate excitotoxicity models, neuroprotection assays

  • For liver models: metabolic stress conditions, nutrient manipulation

Integrating these approaches allows researchers to comprehensively examine GOT1's role in specific cellular contexts and discover tissue-specific functions that may have therapeutic relevance.

What experimental protocols are recommended for investigating the therapeutic potential of recombinant GOT1?

Investigation of recombinant GOT1 (rGOT1) therapeutic potential requires systematic experimental protocols that address production, efficacy, and translational considerations:

• Production and Characterization of rGOT1:

  • Expression in appropriate systems (bacterial, mammalian)

  • Purification and quality control assessments

  • Verification of enzymatic activity compared to native human GOT1

• In Vitro Efficacy Studies:

  • Cell culture models of glutamate excitotoxicity

  • Measurement of glutamate clearance rates and neuroprotection

• Animal Models for Stroke Research:

  • Transient middle cerebral artery occlusion (MCAO) model for ischemic stroke

  • Assessment parameters:

    • Serum GOT activity monitoring (enzymatic assays)

    • Serum and brain glutamate levels (MR spectroscopy)

    • Infarct volume (MRI at multiple time points)

    • Sensorimotor function (behavioral tests)

    • Long-term outcomes (7+ days post-intervention)

• Dosing and Timing Optimization:

  • Dose-response studies comparing various rGOT1 concentrations

  • Combination therapy with oxaloacetate at different ratios

  • Administration timing relative to ischemia onset (up to 2 hours post-onset has shown efficacy)

  • Route of administration optimization

• Translational Considerations:

  • Dose scaling calculations (accounting for species differences in baseline GOT levels)

  • Safety and immunogenicity assessments

  • Pharmacokinetic studies examining enzyme half-life and activity profiles

The current research suggests that combining human rGOT1 with low doses of oxaloacetate may be a particularly successful approach for stroke treatment, with protective effects persisting even when treatment is delayed up to 2 hours after ischemia onset .

How can researchers effectively detect and characterize GOT1 genetic variants in population studies?

Effective detection and characterization of GOT1 genetic variants in population studies require a comprehensive strategy combining genomic, statistical, and functional approaches:

• Study Design Considerations:

  • Population selection accounting for ethnic diversity

  • Sample size calculations based on expected variant frequencies

  • Phenotyping strategy (e.g., AST activity measurements)

• Genotyping and Sequencing Approaches:

  • Genome-wide association studies (GWAS) with dense SNP arrays

  • Targeted sequencing of GOT1 gene and regulatory regions

  • Quality control procedures:

    • Call rate thresholds (>95%)

    • Minor allele frequency filters

    • Hardy-Weinberg Equilibrium checks

• Variant Validation and Characterization:

  • Confirmation by dideoxy sequencing

  • Custom TaqMan SNP Genotyping Assays for specific variants

  • Functional prediction using bioinformatic tools

  • Conservation analysis across species

• Statistical Analysis Methods:

  • Association testing using mixed model analysis for related individuals

  • Adjustment for covariates (age, sex, population structure)

  • Multiple testing correction

• Functional Validation:

  • In vitro expression of wild-type and mutant proteins using cloning techniques

  • Construction of expression vectors (e.g., using HindIII-anchored upstream primers and XbaI-anchored downstream primers)

  • Enzyme activity assays comparing wild-type and variant proteins

As demonstrated in the identification of the c.1165_1167delAAC variant, this multi-step approach enables researchers to move from initial genetic association to functional characterization, providing insights into the biological significance of GOT1 variants .