Recombinant Mouse Carnitine O-palmitoyltransferase 1, muscle isoform (Cpt1b)

What is Cpt1b and what is its functional role in metabolism?

Carnitine palmitoyltransferase 1b (Cpt1b) is the muscle isoform of CPT1, a rate-limiting enzyme in mitochondrial β-oxidation that controls the mitochondrial uptake of long-chain acyl-CoAs. Cpt1b catalyzes the formation of acylcarnitines from long-chain fatty acids, facilitating their transport into mitochondria for oxidation and energy production.

Cpt1b is primarily expressed in tissues with high energy demands, including skeletal muscle and heart. In experimental studies, Cpt1b has been confirmed as the main transferase for long-chain acylcarnitine synthesis, with its expression positively associating with medium and long-chain acylcarnitines (12:0, 14:1, 14:2, 16:0, 16:1, 18:0, 18:1) . The mouse Cpt1b protein consists of 772 amino acids (mature protein 2-773), and recombinant proteins typically include tags such as His-tag for purification purposes .

How should researchers properly store and reconstitute recombinant Cpt1b protein?

Recombinant Cpt1b protein requires careful handling for optimal experimental results. Based on standard protocols:

• Store lyophilized recombinant Cpt1b protein at -20°C/-80°C upon receipt

• Aliquot the protein to avoid repeated freeze-thaw cycles, which can degrade protein activity

• For reconstitution:

  • Briefly centrifuge the vial prior to opening

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

  • Add glycerol (5-50% final concentration) for long-term storage

  • Store working aliquots at 4°C for up to one week

The storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0. The protein should maintain greater than 90% purity as determined by SDS-PAGE .

What are the common methods to detect and measure Cpt1b expression?

Several validated methods are employed to detect and measure Cpt1b expression:

• Quantitative Reverse Transcriptase PCR (qRT-PCR):

  • Primers for mouse Cpt1b: 5'-CCTGCTACATGGCAACTGCTA-3' (sense) and 5'-AGAGGTGCCCAATGATGGGA-3' (antisense)

  • GAPDH (control): 5'-GGAGCGAGATCCCTCCAAAAT-3' (sense) and 5'-GGCTGTTGTCATACTTCTCATGG-3' (antisense)

  • Single-cell qRT-PCR can also be used to analyze Cpt1b expression at different developmental stages

• Western Blotting:

  • Using specific antibodies such as recombinant monoclonal antibodies against Cpt1b

  • The antibodies recognize epitopes specific to muscle isoform Cpt1b

• Cpt1 Activity Assay:

  • Modified mitochondrial Cpt1 assay measuring the rate of formation of palmitoylcarnitine from palmitoyl-CoA plus carnitine

  • Typical assay conditions involve isolated mitochondria or tissue homogenates

How does Cpt1b deficiency affect cardiac function under stress conditions?

Cpt1b deficiency has significant implications for cardiac function, particularly under stress conditions. Studies using heterozygous Cpt1b knockout mice (Cpt1b+/-) have provided crucial insights:

• Under basal conditions:

  • Cpt1b+/- mice show overtly normal cardiac structure and function

  • CPT1 activity is partially reduced (approximately 50%) compared to wild-type mice

• Under pressure-overload conditions (Transverse Aorta Constriction - TAC):

  • Cpt1b+/- mice are significantly more susceptible to premature death with congestive heart failure

  • Exhibit exacerbated cardiac hypertrophy and remodeling

  • Show more pronounced impairments of cardiac contraction

  • Demonstrate greater eccentric cardiac hypertrophy than wild-type littermates

The survival rate under severe pressure-overload conditions shows dramatic differences:

• Wild-type mice: Majority survive two weeks of TAC

• Cpt1b+/- mice: Majority die before the two-week term with signs of heart failure (dilated heart, effluence, shortness of breath)

Echocardiographic measurements after TAC in Cpt1b+/- mice compared to wild-type show:

These findings contradict the common view that fatty acid oxidation depression may be beneficial for the heart in cardiac hypertrophy and heart failure, suggesting that Cpt1b deficiency can cause lipotoxicity under pathological stress .

What is the role of Cpt1b in skeletal muscle metabolism and insulin sensitivity?

Research using muscle-specific Cpt1b knockout mice (Cpt1bm-/-) has revealed complex metabolic adaptations:

• Mitochondrial fatty acid oxidation (FAO):

  • Cpt1bm-/- mice show impaired mitochondrial FAO in skeletal muscle

  • This leads to decreased whole-body adiposity compared to control mice

• Body composition changes:

  • Body weight and fat mass become significantly less in Cpt1bm-/- mice

  • These changes occur before food intake becomes significantly less

  • Fat-free mass only becomes significantly less around 100 days of age

• Insulin sensitivity:

  • Paradoxically, despite lipid accumulation (typically associated with insulin resistance), Cpt1bm-/- mice maintain insulin sensitivity

  • Plasma insulin values remain low in Cpt1bm-/- mice with age, while increasing in control mice

  • Insulin tolerance tests show equivalent insulin response in weight-matched Cpt1bm-/- and control mice at 10-12 weeks and 18-20 weeks of age

These findings challenge both the lipotoxicity hypothesis and the mitochondrial overload hypothesis of insulin resistance, suggesting adaptive mechanisms that preserve insulin sensitivity despite impaired FAO .

How is Cpt1b expression regulated during embryonic development?

Temporal regulation of Cpt1b expression during preimplantation development follows a specific pattern:

• Early embryonic development:

  • Cpt1b transcripts are first detected at the 2-cell stage

  • They subsequently disappear and reappear at the morula and blastocyst stages

  • This timing coincides with increasing profiles of oxygen uptake and fatty acid oxidation

• Oocyte expression patterns:

  • Analysis of single-cell quantitative RT-PCR shows variable Cpt1b expression in antral and ovulated metaphase II (MII) oocytes

  • MII oocytes show two distinct groups of Cpt1b expression levels

  • Antral oocytes, classified by chromatin configuration, show three groups with different numbers of Cpt1b transcripts

This developmental regulation suggests critical roles for Cpt1b in energy metabolism during early embryonic development, particularly during the transition to increased oxygen consumption and fatty acid utilization at the morula stage .

Cpt1b in Acute Myeloid Leukemia (AML)

Studies have shown significant associations between Cpt1b expression and AML:

• Expression levels:

  • Cpt1b is markedly higher in AML patients compared to normal subjects

  • This upregulation is associated with poor clinical outcomes

• Prognostic value:

  • Univariate analysis shows that high Cpt1b expression is associated with poorer survival (HR: 1.526, 95% CI: 1.065-2.187, p=0.021)

  • This remains significant even after accounting for other established prognostic factors

Cpt1b in Placental Function and Maternal Age

Recent research has identified associations between maternal age, placental Cpt1b expression, and metabolic health:

• Age-related changes:

  • Older maternal age associates with lower expression of placental Cpt1b

  • This relationship is not observed with other Cpt isoforms (Cpt1a, Cpt1c, or Cpt2)

• Acylcarnitine synthesis:

  • Cpt1b expression positively associates with eight medium and long-chain acylcarnitines

  • In women with BMI ≥25 kg/m², older maternal age associates with reductions in five acylcarnitines (12:0, 14:1, 16:0, 16:1, 18:1)

  • These associations disappear after adjusting for Cpt1b expression, suggesting Cpt1b mediates the relationship

These findings suggest that age-related Cpt1b decline may underlie decreased placental metabolic flexibility, potentially contributing to pregnancy complications in older women, particularly those with higher BMI .

What experimental models are most suitable for studying Cpt1b function?

Several experimental models have been successfully employed to study Cpt1b function:

• Genetic knockout models:

  • Heterozygous Cpt1b+/- mice: Show approximately 50% reduction in cardiac Cpt1 activity, useful for studying partial inhibition effects

  • Muscle-specific Cpt1bm-/- mice: Generated using Cre-loxP system with muscle creatine kinase promoter, allow tissue-specific analysis

• Recombinant protein systems:

  • Full-length recombinant mouse Cpt1b protein with His-tag expressed in E. coli

  • Provides tools for in vitro enzymatic studies and antibody production

• Cell culture models:

  • Transfected cell lines expressing Cpt1b for metabolic studies

  • Primary cardiomyocytes or myoblasts from Cpt1b knockout mice

• Assay systems:

  • Mitochondrial Cpt1 activity assay measuring palmitoylcarnitine formation

  • LC-MS/MS analysis of acylcarnitines to assess functional outcomes of Cpt1b activity

The choice of model should align with specific research questions, considering whether systemic or tissue-specific effects are of interest, and whether complete or partial inhibition of Cpt1b activity is desired.

How do researchers accurately measure Cpt1b enzymatic activity?

The gold standard approach for measuring Cpt1b enzymatic activity involves:

• Mitochondrial preparation:

  • Isolation of intact mitochondria from tissue samples

  • Careful handling to maintain mitochondrial integrity

• Activity assay:

  • Measurement of palmitoylcarnitine formation from palmitoyl-CoA and carnitine

  • Standardized reaction conditions (temperature, pH, substrate concentrations)

  • Inclusion of appropriate controls to validate specificity

• Detection methods:

  • Radioisotope-based assays using [¹⁴C]-labeled substrates

  • HPLC or LC-MS/MS for direct quantification of palmitoylcarnitine

  • Spectrophotometric coupled enzyme assays

• Data normalization:

  • Expression of activity per unit protein or mitochondrial marker

  • Accounting for tissue-specific differences in expression

Researchers should be aware that Cpt1b activity is sensitive to malonyl-CoA inhibition, which provides physiological regulation of the enzyme, and this sensitivity should be considered when interpreting activity measurements .

What are the key considerations when designing Cpt1b knockout or knockdown studies?

When designing genetic manipulation studies for Cpt1b:

• Choice of knockout strategy:

  • Global knockout: Likely embryonic lethal due to essential metabolic functions

  • Heterozygous knockout (Cpt1b+/-): Allows study of partial deficiency, similar to pharmacological inhibition

  • Tissue-specific knockout: Enables focused study of Cpt1b in specific tissues (heart, skeletal muscle)

  • Inducible knockout: Controls timing of Cpt1b deletion to avoid developmental compensation

• Experimental conditions:

  • Basal vs. stressed conditions: Cpt1b deficiency may only manifest phenotypes under stress (e.g., TAC for cardiac studies)

  • Dietary manipulations: High-fat diet, fasting, or exercise challenges may reveal metabolic phenotypes

• Comprehensive phenotyping:

  • Metabolic profiling: Acylcarnitines, fatty acids, glucose metabolism

  • Tissue function: Cardiac parameters, skeletal muscle performance

  • Mitochondrial assessment: Respiration, morphology, membrane potential

• Controls and validation:

  • Confirmation of Cpt1b deletion at mRNA and protein levels

  • Measurement of Cpt1 enzymatic activity to confirm functional impact

  • Wild-type littermates as controls to minimize genetic background effects

How can researchers effectively analyze the metabolic consequences of altered Cpt1b activity?

A comprehensive metabolic analysis approach includes:

• Acylcarnitine profiling:

  • LC-MS/MS quantification of medium and long-chain acylcarnitines

  • Analysis of tissue extracts and plasma/serum samples

  • Comparison across multiple chain lengths (C12-C18) to assess specific impacts

• Lipid accumulation assessment:

  • Quantification of triglycerides, diacylglycerols, and ceramides in tissues

  • Histological evaluation using Oil Red O or other lipid stains

  • Electron microscopy to assess lipid droplet size and distribution

• Energy metabolism evaluation:

  • Indirect calorimetry to measure whole-body metabolism

  • Glucose tolerance and insulin sensitivity tests

  • Tracer studies to track fatty acid and glucose utilization

  • Respirometry to assess mitochondrial oxygen consumption

• Molecular pathway analysis:

  • Expression of fatty acid metabolism genes

  • Activation status of key signaling pathways (AMPK, mTOR, insulin signaling)

  • Mitochondrial biogenesis and quality control markers

By combining these approaches, researchers can gain comprehensive insights into how Cpt1b activity impacts cellular and organismal metabolism.

What are the emerging areas of Cpt1b research with therapeutic potential?

Several promising research directions are developing:

• Targeted Cpt1b modulation:

  • Tissue-specific inhibitors to avoid systemic effects

  • Temporal control of inhibition to match physiological needs

  • Understanding the therapeutic window for partial Cpt1b inhibition

• Disease-specific applications:

  • Heart failure: Resolving contradictions between apparent benefits of Cpt1 inhibitors and detrimental effects seen in Cpt1b+/- mice

  • Cancer metabolism: Exploring the role of Cpt1b upregulation in AML and potential for targeted therapy

  • Metabolic diseases: Investigating adaptive responses to impaired FAO and implications for insulin sensitivity

• Developmental biology:

  • Further characterization of Cpt1b's role in embryonic development

  • Understanding maternal-fetal metabolic interactions involving Cpt1b

• Aging research:

  • Exploring the mechanisms of age-related Cpt1b decline in various tissues

  • Interventions to maintain Cpt1b function during aging

How do conflicting findings regarding Cpt1b function inform research design?

The literature contains apparent contradictions regarding Cpt1b function and inhibition:

• Cardiac effects contradiction:

  • Clinical trials show benefits of Cpt1 inhibitors for cardiac hypertrophy and heart failure

  • Genetic models show Cpt1b deficiency aggravates cardiac pathology under stress

• Metabolic effects contradiction:

  • Decreased FAO is associated with lipotoxicity and insulin resistance in many models

  • Cpt1bm-/- mice show decreased FAO but preserved insulin sensitivity

These contradictions inform research design considerations:

• Dose effects: Partial vs. complete inhibition may have different outcomes

• Timing: Acute vs. chronic inhibition may trigger different adaptive responses

• Tissue specificity: Effects may differ based on tissue-specific metabolic requirements

• Compensatory mechanisms: Chronic genetic deficiency may allow adaptation that acute pharmacological inhibition does not

• Experimental context: Stress conditions, dietary factors, and genetic background may influence outcomes