Recombinant Mouse Carnitine O-palmitoyltransferase 1, liver isoform (Cpt1a)

What is the functional role of CPT1a in fatty acid metabolism?

The enzyme function can be summarized in the following reaction:
$\text{Long-chain acyl-CoA} + \text{Carnitine} \xrightarrow{\text{CPT1a}} \text{Long-chain acylcarnitine} + \text{CoA}$

This reaction is particularly important under conditions of increased energy demand or low glucose availability, when fatty acid oxidation becomes a primary source of cellular energy.

How do mouse CPT1a expression patterns differ across tissues?

Mouse CPT1a shows distinct tissue-specific expression patterns that differ from other CPT1 isoforms:

This tissue-specific expression contributes to the metabolic specialization of different organs. Interestingly, CPT1a mRNA is expressed at about 0.3% of the level in BAT in mouse placenta, whereas CPT1a is the predominant isoform expressed in placenta compared to CPT1b .

What methodologies are available for measuring CPT1a activity?

Several complementary approaches can be used to assess CPT1a activity in research settings:

• Radiochemical forward assay: This method measures the formation of acylcarnitines using radiolabeled substrates, providing high sensitivity and specificity. For implementation, tissue homogenates are prepared in buffer (typically 10 mM potassium phosphate/150 mM NaCl, pH 7.4) supplemented with protease inhibitor cocktail and protein phosphatase inhibitors .

• Colorimetric CoA detection assay: This method employs DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) to detect free CoA released during CPT1 catalysis. The reaction between free thiols from CoA and DTNB produces 2-thio-5-nitrobenzoic acid (TNB), which can be measured at 412 nm. This approach is more amenable to high-throughput screening than radioisotope methods .

• ELISA-based detection: Commercial ELISA kits with high sensitivity (e.g., 39.2 pg/mL) and detection ranges of 78-5000 pg/ml are available for quantifying CPT1a protein levels, though they do not directly measure enzymatic activity .

• GC/MS isotope-ratio approach: This method can be used to measure intracellular metabolites related to CPT1a activity, providing insights into the enzyme's impact on cellular metabolism .

Each method offers advantages depending on the specific research question, available equipment, and required throughput.

What expression systems are most effective for producing active recombinant mouse CPT1a?

Two primary expression systems have demonstrated success in producing functional recombinant mouse CPT1a:

• E. coli expression system: Recombinant full-length mouse CPT1a protein (P97742, amino acids 2-773) with an N-terminal His tag can be effectively expressed in E. coli. This approach typically yields protein with >90% purity as determined by SDS-PAGE . While this system provides high protein yields, the bacterial expression environment lacks mammalian post-translational modifications.

• Mammalian expression systems: Direct expression of CPT1a in Expi293 cells provides a reliable source of catalytically active CPT1a. The mitochondrial extracts isolated from in-vitro cell culture represent a scalable source of functional enzyme. This system allows for proper protein folding and mammalian-specific post-translational modifications that may be critical for certain CPT1a functions .

When selecting an expression system, researchers should consider:

• The need for post-translational modifications

• Required protein yield and purity

• Downstream application requirements (structural studies vs. activity assays)

• Whether membrane association is necessary for the experimental design

How can researchers validate CPT1a enzymatic activity in experimental settings?

Multiple complementary approaches should be employed to validate CPT1a enzymatic activity:

• Substrate-dependent activity assays: Demonstrate that activity requires both palmitoyl-CoA and carnitine, the physiological substrates of CPT1a.

• Colorimetric CoA detection: Measure the release of CoA during the reaction using DTNB, which produces a yellow-colored product (TNB) detectable at 412 nm. This method can be scaled to 96-well format for higher throughput screening .

• Inhibitor profiling: Validate activity using dose-dependent inhibition with known CPT1a inhibitors such as etomoxir, perhexiline, and chlorpromazine. A properly functioning CPT1a preparation should show expected IC50 values for these reference compounds .

• Kinetic parameter determination: Calculate Km values for both substrates (palmitoyl-CoA and carnitine) and compare with literature values for native enzyme.

• Isoform-specific verification: Ensure that the measured activity is specifically from CPT1a rather than other CPT isoforms, particularly when working with tissue extracts containing multiple isoforms.

A comprehensive validation protocol should include positive and negative controls, concentration-dependent effects, and appropriate statistical analysis of replicate measurements.

What are the critical storage and handling parameters for recombinant CPT1a?

To maintain optimal activity of recombinant mouse CPT1a:

• Storage conditions:

  • Store at -20°C to -80°C upon receipt

  • Aliquot to avoid repeated freeze-thaw cycles, which can significantly reduce activity

  • Working aliquots can be stored at 4°C for up to one week

• Reconstitution protocol:

  • Briefly centrifuge the vial prior to opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • For long-term storage, add glycerol to a final concentration of 5-50% (typically 50%)

• Buffer composition:

  • Typically maintained in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  • Protease inhibitors should be added when working with enzyme preparations

• Stability considerations:

  • CPT1a is a membrane-associated enzyme sensitive to detergents

  • CPT1 isozymes are located in the mitochondrial outer membrane and are detergent-labile

  • Activity may be affected by freeze-thaw cycles, so single-use aliquots are recommended

Proper storage and handling protocols are essential for maintaining the structural integrity and enzymatic activity of recombinant CPT1a.

How does the G710E mutation affect CPT1a function and experimental applications?

The G710E mutation in CPT1a provides a valuable tool for studying the enzyme's dual functionality. This mutation selectively inactivates the canonical carnitine palmitoyltransferase (CPTase) activity while preserving its lysine succinyltransferase (LSTase) activity .

Key functional differences between wild-type and G710E mutant CPT1a:

This mutation enables researchers to:

• Dissect the specific roles of CPTase versus LSTase activities in metabolic regulation

• Study protein succinylation effects independent of fatty acid oxidation

• Investigate the metabolic consequences of CPT1a-mediated protein modification

• Examine non-canonical functions of CPT1a in cellular metabolism

The G710E mutant represents an elegant approach to separate the dual functionalities of CPT1a, allowing for more precise mechanistic studies of this multifunctional enzyme .

What experimental approaches can reveal CPT1a's dual enzymatic functions?

To investigate CPT1a's roles as both a carnitine palmitoyltransferase and a lysine succinyltransferase, researchers can employ several complementary strategies:

• Mutational analysis: Utilize the G710E mutation that selectively inactivates CPTase activity while preserving LSTase activity, allowing separation of these two functions .

• SILAC-based quantitative proteomics: This approach can identify proteins that are succinylated in a CPT1a-dependent manner. Previous research identified 171 lysine sites on 101 proteins (31% of the total 550 detected unique lysine sites) that showed increased succinylation by more than 1.5-fold in cells expressing wild-type CPT1a compared to control cells .

• Parallel activity assays:

  • For CPTase: Measure the formation of acylcarnitines from palmitoyl-CoA and carnitine

  • For LSTase: Assess the succinylation of target proteins such as enolase 1

• Target protein functional analysis: Examine how CPT1a-mediated succinylation affects the enzymatic activity or other functions of target proteins. For example, CPT1a succinylates enolase 1 and reduces its enzymatic activity both in cells and in vitro .

• Metabolic consequence assessment: Evaluate the physiological outcomes of each activity under specific conditions, such as glutamine depletion, which revealed a role for the LSTase activity in promoting cell proliferation .

By combining these approaches, researchers can build a comprehensive understanding of how CPT1a's dual enzymatic activities contribute to cellular metabolism in both normal and pathological states.

What are the metabolic consequences of CPT1a inhibition or deletion?

Inhibition or genetic deletion of CPT1a produces complex and sometimes counterintuitive metabolic effects:

• Liver-specific effects: Hepatocyte-specific Cpt1a knockout (LKO) mice display more severe hepatosteatosis (fatty liver) when fed a high-fat diet, yet are paradoxically protected against:

  • Diet-induced weight gain

  • Insulin resistance

  • Hepatic endoplasmic reticulum stress

  • Liver inflammation and damage

• Systemic metabolic benefits: LKO mice show:

  • Increased energy expenditure

  • Enhanced adipose tissue browning

  • Activation of the PPARα-FGF21 axis in the liver

• FGF21-mediated cross-talk: The metabolic benefits of hepatic CPT1a deletion are dependent on FGF21, as antibody-mediated neutralization of FGF21 abolished the improved metabolic phenotype and adipose browning in LKO mice .

• Therapeutic implications: These findings suggest that targeted inhibition of hepatic CPT1a might represent a viable strategy for treating obesity and non-alcoholic fatty liver disease (NAFLD), despite promoting hepatic steatosis .

These observations highlight the complexity of metabolic regulation and the importance of tissue-specific and context-dependent effects when modulating CPT1a activity for research or therapeutic purposes.

How should researchers design experiments to study CPT1a-mediated liver-adipose cross-talk?

To effectively investigate CPT1a's role in inter-organ communication, particularly between liver and adipose tissue, several key experimental design elements should be considered:

• Tissue-specific genetic models: Utilize hepatocyte-specific Cpt1a knockout mice (as in the LKO model) to isolate liver-specific effects from systemic consequences .

• Cross-tissue sampling: Collect and analyze samples from multiple relevant tissues (liver, brown adipose tissue, white adipose tissue) to capture the full scope of metabolic adaptations.

• Mediator identification and validation:

  • Perform RNA sequencing to identify potential signaling molecules (e.g., FGF21)

  • Validate their role through neutralization experiments (e.g., anti-FGF21 antibodies)

  • Measure circulating levels of identified mediators

• Environmental challenges: Test the cross-talk under various conditions:

  • Dietary interventions (high-fat diet)

  • Temperature challenges (cold exposure)

  • Fasting-refeeding cycles

• Temporal analyses: Examine the time course of changes in both liver and adipose tissues to establish cause-and-effect relationships.

• Sex-specific considerations: Design experiments to account for sex differences, as CPT isoform expression can vary significantly between male and female mice (e.g., CPT1a in white adipose tissue of male but not female mice; CPT1b in white adipose tissue of female but not male mice) .

• Functional readouts: Include relevant functional assessments:

  • Metabolic cage studies for energy expenditure

  • Body temperature monitoring

  • Glucose tolerance tests

  • Insulin sensitivity measurements

  • Histological analysis of both liver and adipose tissues

This comprehensive experimental approach will help elucidate the complex metabolic interplay mediated by CPT1a across multiple tissues.

How can apparent discrepancies between in vitro and in vivo CPT1a activity data be reconciled?

When faced with discrepancies between in vitro and in vivo CPT1a activity data, researchers should consider several potential explanations:

• Membrane environment effects: CPT1a is located in the outer mitochondrial membrane and is detergent-labile . In vitro assays may not fully recapitulate this native membrane environment, affecting enzyme conformation and activity.

• Endogenous regulators: Natural inhibitors such as malonyl-CoA and succinyl-CoA regulate CPT1a activity in vivo. Search result 2 notes that "short-chain dicarboxylic-acyl-CoAs, including succinyl-CoA, bind to carnitine palmitoyltransferase 1A (CPT1A) and inhibit its carnitine palmitoyltransferase (CPTase) activity" . These regulators may be present at different concentrations in various experimental settings.

• Multi-organ effects: In vivo, CPT1a function in one tissue affects others through hormonal mediators like FGF21 . Such cross-talk cannot be observed in isolated in vitro systems.

• Isoform compensation: Other CPT isoforms may compensate for CPT1a modulation in vivo. For example, in CPT1b+/− mice, while skeletal muscle CPT-1 activity decreased to ~60%, there was "no change in total liver CPT-1 activity" .

• Dual enzymatic activities: CPT1a functions as both a CPTase and an LSTase. The relative contribution of each activity may differ between in vitro and in vivo settings .

To address these discrepancies, researchers should:

• Validate findings across multiple experimental systems

• Use tissue-specific genetic models

• Assess both direct enzymatic activities and indirect physiological consequences

• Consider the broader metabolic and signaling context

• Develop more physiologically relevant in vitro systems

How should unexpected results in CPT1a inhibition or knockout studies be interpreted?

When confronted with unexpected outcomes from CPT1a inhibition or knockout studies, researchers should systematically evaluate several possible explanations:

• Dual enzyme functionality: Consider which of CPT1a's enzymatic activities (CPTase vs. LSTase) is affected by the intervention. The G710E mutation demonstrated that these activities can be separated and may contribute differently to metabolic outcomes .

• Hormonal mediation: Evaluate whether the effects are mediated by hormones or signaling molecules. For example, the paradoxical metabolic benefits of hepatic CPT1a deletion were found to be FGF21-dependent .

• Compensatory mechanisms: Assess whether other enzymes or pathways are upregulated to compensate for CPT1a loss. This could include other CPT isoforms or alternative metabolic pathways.

• Tissue-specific versus systemic effects: Distinguish between local effects in the tissue where CPT1a is modulated and systemic consequences. Hepatic CPT1a deletion caused increased liver steatosis but beneficial effects on adipose tissue browning and energy expenditure .

• Temporal considerations: Determine whether the observed effects are immediate consequences of CPT1a modulation or represent longer-term adaptations.

• Experimental conditions: Consider whether the specific conditions (diet, temperature, stress) influence the outcome of CPT1a modulation. Some effects may only become apparent under metabolic challenge.

• Technical validation: Confirm the specificity and extent of CPT1a inhibition or knockout, ensuring the observed effects are truly attributable to CPT1a modulation.

A comprehensive analysis that considers these factors will help researchers interpret unexpected results and potentially uncover novel insights into CPT1a's role in metabolism.

How can CPT1a research contribute to therapeutic approaches for metabolic diseases?

CPT1a research offers several promising avenues for metabolic disease therapeutics:

• Obesity treatment: Hepatic CPT1a inhibition has shown potential in treating obesity by increasing energy expenditure and promoting adipose tissue browning through an FGF21-dependent mechanism .

• NAFLD management: Despite causing increased hepatic steatosis, liver-specific CPT1a deletion protects against high-fat diet-induced liver damage, inflammation, and insulin resistance, suggesting a potential approach for treating non-alcoholic fatty liver disease .

• Diabetes therapy: By modulating hepatic lipid metabolism and improving whole-body insulin sensitivity, CPT1a inhibition might contribute to diabetes management strategies.

• Cancer metabolism targeting: The dual functionality of CPT1a (particularly its LSTase activity) provides potential targets for cancer therapies, as this activity has been shown to regulate cell proliferation under specific metabolic conditions .

• Biomarker development: Understanding CPT1a's role in different pathological states may lead to the development of diagnostic or prognostic biomarkers for metabolic disorders.

The paradoxical finding that hepatic CPT1a inhibition can induce a "healthy steatotic status" highlights the complexity of metabolic regulation and suggests that targeted, tissue-specific modulation of CPT1a activity could offer therapeutic benefits despite seemingly contradictory effects on lipid accumulation .

What technological advances are improving CPT1a research capabilities?

Recent technological developments have expanded the toolkit for CPT1a research:

• High-throughput activity assays: Modified colorimetric CoA detection methods have been optimized for 96-well format screening, enabling more efficient identification of CPT1a modulators without the need for radioisotopes .

• Direct expression systems: Systems using Expi293 cells transfected with CPT1a plasmid provide a reliable and robust source of catalytically active human CPT1a without requiring protein purification .

• SILAC-based proteomics: This approach enables identification of proteins succinylated by CPT1a's LSTase activity, revealing 171 lysine sites on 101 proteins modified in a CPT1a-dependent manner .

• Selective mutations: Mutations like G710E that selectively inactivate one function while preserving another allow for precise dissection of CPT1a's dual enzymatic roles .

• Tissue-specific knockout models: Hepatocyte-specific Cpt1a knockout mice have revealed unexpected aspects of liver-adipose cross-talk and metabolic regulation .

• Sensitive ELISA detection: Commercial ELISA kits with sensitivity as low as 39.2 pg/mL allow precise quantification of CPT1a protein in various sample types .

These advances provide researchers with more precise tools to investigate CPT1a's complex roles in metabolism, potentially accelerating the development of therapeutic approaches for metabolic disorders.