Recombinant Mouse Serpin A12 (Serpina12)

What is Recombinant Mouse Serpin A12 and how does it function in metabolic regulation?

Recombinant Mouse Serpin A12, also known as Vaspin, is a 45-50 kDa secreted adipokine that belongs to the serpin family of serine protease inhibitors . Its primary function involves maintaining insulin sensitivity by circulating in a complex with Kallikrein 7 (KLK7) and preventing KLK7-mediated cleavage of insulin . Serpin A12 exhibits multiple metabolic functions: it promotes elevation of circulating insulin levels, improves glucose tolerance, and can inhibit high glucose-induced activation of the insulin receptor .

Beyond metabolic regulation, Serpin A12 inhibits TRANCE/RANK L-induced osteoclast development and inflammatory activation of vascular smooth muscle and endothelial cells . The protein consists of 414 amino acids and functions primarily by modulating the insulin-inhibiting protease KLK7 in adipose tissue .

Where is Serpin A12 primarily expressed in mice and how does expression vary by tissue type?

Serpin A12 expression in mice occurs across multiple tissues with varying levels of expression. The primary sites of expression include:

• Adipose tissue (both visceral and subcutaneous adipocytes)

• Brown adipose tissue (BAT)

• Gastric glands and epithelium

• Placenta

• Skin

• Stomach

• Liver

Expression analysis using quantitative PCR reveals that endogenous mouse vaspin (serpinA12) is highly expressed in skin, stomach, liver, and brown adipose tissue . In transgenic models expressing human SERPINA12, the highest expression typically occurs in BAT, followed by subcutaneous (inguinal) white adipose tissue (iWAT), with lower expression in epididymal white adipose tissue (eWAT) .

How do Serpin A12 levels correlate with metabolic conditions in experimental models?

Serpin A12 levels demonstrate significant correlation with metabolic status in both rodent models and humans. In the Otsuka Long Evans Tokushima Fatty (OLETF) rat model, Serpin A12 expression peaks concurrently with obesity and plasma insulin levels at approximately 30 weeks of age . Similarly, in humans, serum vaspin levels are elevated in individuals with obesity and type 2 diabetes .

In experimental mouse models, administration of recombinant vaspin to obese, insulin-resistant mice counteracts obesity-induced inflammation and dysfunction in both adipose tissue and liver, partially restoring glucose tolerance . Metabolic health of mice overexpressing vaspin demonstrates greater resistance to obesogenic conditions, while vaspin knockout exacerbates metabolic dysfunction in obesity, establishing vaspin as a beneficial and compensatory factor in obesity-related disorders .

What are the optimal procedures for measuring Serpin A12 inhibitory activity in vitro?

Serpin A12 inhibitory activity can be precisely measured using a fluorogenic substrate assay that assesses its ability to inhibit Kallikrein 7 (KLK7) activity. The standardized protocol involves:

• Preparation of reagents:

  • Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5

  • Inhibition Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5

  • Assay Buffer: 50 mM Tris, 150 mM NaCl, pH 8.5

  • Recombinant Mouse Serpin A12

  • Recombinant Human Kallikrein 7 (rhKLK7)

  • Fluorogenic substrate: Mca-RPKPVE-Nval-WRK(Dnp)-NH₂

• Assay procedure:

  • Prepare a Serpin A12 dilution series (neat/50, 160, 80, 40, 20, 10, 5, 2, and 0.5 nM)

  • Combine equal volumes of each Serpin A12 dilution with 50 μg/mL rhKLK7

  • Include an enzyme control containing equal volumes of Inhibition Buffer and 50 μg/mL rhKLK7

  • Incubate reaction mixtures at 37°C for 30 minutes

  • Dilute each reaction 12.5-fold using Assay Buffer

  • Load 50 μL of diluted reactions to a plate, and add 50 μL of 20 μM substrate

  • Include a substrate blank containing assay buffer and substrate

  • Measure fluorescence at excitation/emission wavelengths of 320/405 nm for 5 minutes

• Data analysis:

  • Calculate specific activity using the formula:
    Specific Activity (pmol/min/μg) = [Adjusted Vmax (RFU/min) × Conversion Factor (pmol/RFU)] / amount of enzyme (μg)

  • Determine the IC₅₀ value by plotting RFU/min versus Serpin A12 concentration using 4-parameter logistic fitting

  • Functional Serpin A12 should demonstrate an IC₅₀ of <60 nM under these conditions

This standardized protocol enables quantitative assessment of Serpin A12's inhibitory capacity against its target protease.

What are key design considerations for experiments using transgenic Serpin A12 mouse models?

When designing experiments with transgenic Serpin A12 mouse models, several critical factors must be addressed to ensure experimental validity and interpretable results:

• Expression validation:

  • Confirm transgene expression through qPCR using primers specific to human SERPINA12 (for human transgene models)

  • Quantify circulating protein levels using species-specific ELISAs

  • Compare expression levels to physiological ranges - transgenic models may express supraphysiological levels (>200 ng/ml compared to normal levels)

• Genetic background considerations:

  • Perform sufficient backcrossing (minimum 5 generations) to establish a pure genetic background

  • Use appropriate littermate controls rather than separate wild-type colonies

  • Consider that the C57Bl/6N background is commonly used for metabolic studies

• Promoter selection:

  • The choice of promoter determines tissue specificity of expression (e.g., aP2 promoter directs expression to adipose tissue)

  • Document complete tissue expression patterns including potential ectopic expression

• Comprehensive phenotyping:

  • Metabolic parameters: glucose tolerance tests, insulin levels, lipid profiles

  • Tissue analysis: adipose tissue and liver histology to assess tissue remodeling

  • Energy homeostasis: food intake, energy expenditure, physical activity

  • Inflammatory markers: cytokine profiles in relevant tissues

  • Both sexes should be analyzed as metabolic phenotypes often show sexual dimorphism

• Environmental challenges:

  • Test both standard chow and high-fat diet conditions to reveal phenotypes that may only manifest under metabolic stress

  • Consider additional challenges such as glucose/insulin tolerance tests, cold exposure, or fasting/refeeding protocols

These design considerations ensure robust experimental protocols for investigating Serpin A12's functions using transgenic models.

What are the recommended methods for quantifying mouse Serpin A12 expression in different tissues?

Accurate quantification of mouse Serpin A12 expression requires tissue-specific approaches and careful methodology:

• mRNA expression analysis by quantitative PCR:

  • RNA isolation: Extract total RNA from tissues using TRIzol followed by RNA cleanup (RNeasy MinElute)

  • Reverse transcription: Convert 500 ng RNA to cDNA using standard protocols

  • qPCR assays: Use TaqMan methodology with species-specific primers

    • Mouse endogenous serpinA12: Mm00471557_m1

    • Human SERPINA12 (for transgenic models): Hs00545180_m1

  • Data normalization: Calculate expression using the ΔΔCT method normalized to housekeeping genes (e.g., 36b4)

• Protein quantification:

  • Tissue protein extraction: Homogenize tissues in appropriate lysis buffers with protease inhibitors

  • Western blotting: Separate proteins by SDS-PAGE, transfer to membranes, and probe with specific anti-Serpin A12 antibodies

  • ELISA: Use commercial or custom ELISAs for serum/plasma quantification, ensuring species specificity

  • Immunohistochemistry: Visualize tissue distribution using specific antibodies on fixed tissue sections

• Tissue-specific considerations:

  • Adipose tissue requires careful handling to minimize degradation; flash freezing is recommended

  • Liver samples should be collected consistently relative to feeding state due to metabolic fluctuations

  • Expression in multiple adipose depots (subcutaneous, visceral, brown) should be analyzed separately

• Experimental controls:

  • Include tissues from knockout mice as negative controls

  • For transgenic models, distinguish between endogenous mouse serpinA12 and transgenic human SERPINA12

  • Standardize collection time and nutritional status across experimental groups

These methods provide comprehensive approaches for accurate quantification of Serpin A12 expression across multiple tissues and experimental conditions.

How can Recombinant Mouse Serpin A12 be used to investigate insulin resistance mechanisms?

Recombinant Mouse Serpin A12 provides a valuable tool for investigating insulin resistance mechanisms through several sophisticated experimental approaches:

• In vivo administration studies:

  • Administer purified recombinant Serpin A12 to diet-induced or genetically obese mice

  • Assess improvements in glucose tolerance and insulin sensitivity through:

    • Glucose tolerance tests (GTT) and insulin tolerance tests (ITT)

    • Hyperinsulinemic-euglycemic clamp studies for tissue-specific insulin sensitivity

    • Phosphorylation status of insulin signaling proteins (insulin receptor, IRS-1, Akt) in multiple tissues

• Mechanistic pathway analysis:

  • Investigate Serpin A12's protection of insulin from KLK7-mediated degradation

  • Examine effects on insulin receptor activation under high glucose conditions

  • Analyze inflammatory signaling pathways that may mediate insulin resistance

  • Study adipokine secretion profiles in the presence of Serpin A12

• Cell-based models:

  • Primary adipocyte cultures treated with Serpin A12 followed by insulin signaling assessment

  • Hepatocyte insulin resistance models to evaluate direct hepatic effects

  • Skeletal muscle cell systems to investigate tissue-specific responses

  • Co-culture systems to examine cross-talk between different metabolic tissues

• Comparative studies with other insulin-sensitizing agents:

  • Side-by-side comparison with established insulin sensitizers (thiazolidinediones, metformin)

  • Combination treatments to investigate potential synergistic effects

  • Assessment of tissue-specific versus systemic effects compared to other agents

These approaches leverage Serpin A12's demonstrated ability to improve glucose tolerance, promote circulating insulin elevation, and counteract obesity-induced tissue dysfunction to provide insights into insulin resistance mechanisms .

What techniques can be employed to study the interaction between Serpin A12 and its target proteases?

Investigating the interaction between Serpin A12 and its target proteases, particularly Kallikrein 7 (KLK7), requires specialized techniques:

• Enzyme inhibition kinetics:

  • Determine inhibition constants (Ki) through steady-state kinetic analysis

  • Characterize inhibition mechanisms (competitive, non-competitive, uncompetitive)

  • Use fluorogenic substrates like Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ to measure protease activity

  • Calculate IC₅₀ values through concentration-response curves and 4-parameter logistic fitting

• Structural biology approaches:

  • X-ray crystallography of Serpin A12-protease complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Molecular modeling based on known serpin-protease structures

  • Site-directed mutagenesis of predicted interaction residues followed by functional testing

• Biophysical interaction analysis:

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinity

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Analytical ultracentrifugation to characterize complex formation

  • Microscale thermophoresis for binding studies in solution

• In situ interaction studies:

  • Proximity ligation assays in tissue sections to visualize interactions

  • FRET-based assays in cellular systems

  • Co-immunoprecipitation from tissues or cell systems expressing both proteins

  • Live-cell imaging with fluorescently tagged proteins

• Proteomic approaches:

  • Mass spectrometry identification of Serpin A12-bound proteins from tissue extracts

  • Crosslinking mass spectrometry to capture transient interactions

  • Serpin A12 affinity purification followed by proteomic analysis of binding partners

These techniques can help elucidate the molecular mechanisms by which Serpin A12 inhibits its target proteases and provides metabolic benefits.

How can researchers use Serpin A12 to investigate adipose tissue-liver cross-talk in metabolic disease?

Serpin A12 provides an excellent model for investigating adipose tissue-liver cross-talk in metabolic disease through several experimental approaches:

• Tissue-specific expression manipulation:

  • Compare adipose-specific versus liver-specific Serpin A12 overexpression/knockdown

  • Assess metabolic parameters in both tissues after manipulation in only one tissue

  • Measure circulating Serpin A12 levels to determine contribution to systemic pool

• Conditioned media experiments:

  • Culture adipose tissue explants from wild-type and Serpin A12-overexpressing mice

  • Collect conditioned media and apply to primary hepatocytes

  • Evaluate changes in hepatocyte insulin sensitivity, lipid metabolism, and inflammatory markers

  • Perform proteomic analysis of secreted factors in conditioned media

• In vivo intervention studies:

  • Administer recombinant Serpin A12 to obese mice

  • Analyze concurrent changes in both adipose tissue and liver

  • Determine if adipose tissue inflammation reduction precedes improvements in liver metabolism

  • Measure tissue-specific glucose uptake and metabolism using labeled glucose tracers

• Molecular signaling analysis:

  • Identify common signaling pathways affected by Serpin A12 in both tissues

  • Investigate whether Serpin A12 directly affects liver or acts through adipokine-mediated signals

  • Examine the impact on inflammatory mediators that might link tissue dysfunction

Existing research demonstrates that Serpin A12 treatment counteracts obesity-induced inflammation and dysfunction in both adipose tissue and liver, suggesting it serves as an important mediator in adipose-liver communication . Transgenic mice overexpressing vaspin show resistance to high-fat diet-induced obesity and metabolic dysfunction, providing evidence for Serpin A12's role in coordinating multi-tissue metabolic responses .

What factors might affect the reproducibility of Serpin A12 functional assays?

Several critical factors can impact reproducibility in Serpin A12 functional assays, requiring careful experimental design and standardization:

• Protein quality factors:

  • Source variation: Recombinant protein production systems (bacterial vs. insect cell-derived) affect post-translational modifications and activity

  • Storage conditions: Multiple freeze-thaw cycles reduce activity; aliquoting and proper storage at -80°C is essential

  • Buffer composition: pH, salt concentration, and presence of stabilizing agents impact protein stability

  • Purity levels: Contaminants may interfere with functional assays

• Assay technical considerations:

  • Temperature control: Enzyme kinetics are highly temperature-dependent

  • Incubation times: Standardization is critical for inhibition assays

  • Substrate quality and concentration: Fluorogenic substrates can degrade with improper storage

  • Instrumentation calibration: Regular calibration of plate readers is essential for fluorescence detection

• Experimental design factors:

  • Appropriate controls: Include enzyme-only, substrate-only, and known inhibitor controls

  • Standard curves: Establish standard curves with each experiment

  • Technical replicates: Minimum triplicate measurements

  • Concentration ranges: Use appropriate dilution series spanning the expected IC₅₀ (0.5-160 nM)

• Data analysis considerations:

  • Curve fitting methods: 4-parameter logistic regression is recommended for IC₅₀ determination

  • Background subtraction: Account for substrate blank readings

  • Reaction rate calculation: Use linear portion of reaction progress curves

  • Normalization approaches: Standardize to positive controls

To optimize reproducibility, researchers should follow standardized protocols as described in the literature, including specific buffer compositions (e.g., 50 mM Tris, 150 mM NaCl, pH 8.5 for assay buffer) and validated substrate concentrations (10 μM) .

How should researchers interpret discrepancies between in vitro and in vivo effects of Serpin A12?

When reconciling discrepancies between in vitro and in vivo effects of Serpin A12, researchers should consider several key factors:

• Physiological context differences:

  • Concentration disparities: In vitro studies often use higher concentrations than physiologically relevant in vivo levels

  • Temporal factors: Acute effects in vitro versus chronic adaptations in vivo

  • Complex environment: In vivo systems include multiple cell types, circulatory factors, and compensatory mechanisms

  • Tissue cross-talk: Effects in one tissue may indirectly impact other tissues in vivo

• Methodological considerations:

  • Model system limitations: Cell lines may not fully recapitulate primary cell behavior

  • Species differences: Mouse and human Serpin A12 share only 61% amino acid sequence identity

  • Technical artifacts: Cell culture conditions may alter cellular phenotypes

  • Readout selection: Different parameters measured in vitro versus in vivo

• Reconciliation strategies:

  • Bridge models: Use ex vivo tissue explants as intermediate complexity systems

  • Concentration-response relationships: Test physiologically relevant concentrations in vitro

  • Time-course studies: Compare acute versus chronic exposures

  • Multi-parameter assessment: Measure identical endpoints in both systems

Research demonstrates that while in vitro studies show direct effects of Serpin A12 on specific cell types, in vivo studies reveal integrated systemic responses. For example, transgenic mice with extremely high circulating Serpin A12 levels (>200 ng/ml) show resistance to diet-induced obesity and enhanced energy expenditure , which may involve complex physiological adaptations beyond what can be observed in cellular models.

What controls and validation steps are essential when studying transgenic Serpin A12 mouse models?

Rigorous experimental design for transgenic Serpin A12 mouse models requires comprehensive controls and validation:

• Genetic validation:

  • Confirmation of transgene integration by PCR and Southern blot analysis

  • Verification of transgene copy number

  • Sequencing to confirm transgene integrity

  • Genotyping protocols with appropriate positive and negative controls

• Expression validation:

  • Quantitative PCR using species-specific primers:

    • Human SERPINA12 (transgene): Hs00545180_m1

    • Mouse serpinA12 (endogenous): Mm00471557_m1

  • Protein expression confirmation by Western blot

  • Serum protein quantification by ELISA

  • Tissue-specific expression analysis across multiple tissues

• Phenotypic characterization controls:

  • Littermate controls matched for age, sex, and housing conditions

  • Wild-type controls from the same genetic background

  • Multiple independent founder lines to control for integration site effects

  • Assessment of potential off-target effects or developmental compensation

• Experimental design considerations:

  • Age-matched groups (metabolic parameters change with age)

  • Sex-balanced cohorts (metabolic phenotypes often show sexual dimorphism)

  • Standardized diet composition and feeding protocols

  • Consistent environmental conditions (temperature, light cycles)

  • Blinded analysis of outcomes where possible

• Functional validation:

  • Verify biological activity of the expressed protein

  • Confirm expected downstream molecular effects

  • Compare phenotype with pharmacological administration of recombinant protein

  • Test multiple metabolic challenges (high-fat diet, glucose tolerance, insulin tolerance)

The h-vaspinTG transgenic model described in the literature underwent rigorous validation, including Southern blot analysis of genomic DNA, serum protein quantification, and tissue-specific expression analysis confirming highest expression in brown adipose tissue followed by subcutaneous white adipose tissue .

How is Serpin A12 research advancing our understanding of obesity and insulin resistance mechanisms?

Serpin A12 research is significantly advancing our understanding of obesity and insulin resistance through several key research directions:

• Protection against metabolic dysfunction:

  • Transgenic models demonstrate that Serpin A12 overexpression provides protection against diet-induced obesity and metabolic dysfunction

  • These findings suggest Serpin A12 acts as a compensatory mechanism during metabolic stress

  • Research indicates that Serpin A12 knockout exacerbates metabolic dysfunction in obesity, further supporting its protective role

• Mechanistic insights:

  • The inhibition of KLK7-mediated insulin degradation represents a novel mechanism influencing insulin bioavailability

  • Serpin A12's ability to improve glucose tolerance while potentially inhibiting high glucose-induced insulin receptor activation suggests complex regulatory functions

  • Anti-inflammatory effects in adipose tissue and vascular cells indicate broader roles beyond direct insulin signaling

• Tissue-specific effects:

  • Expression primarily in adipose tissue with effects on liver function highlights Serpin A12's role in inter-tissue communication

  • Different expression patterns across adipose depots (BAT, subcutaneous vs. visceral) suggest depot-specific functions

  • Effects on both metabolism and inflammation connect these often-linked pathological processes

• Translational potential:

  • Correlation between serum levels and metabolic status in both rodents and humans suggests potential as a biomarker

  • Therapeutic effects of recombinant protein administration provide proof-of-concept for intervention strategies

  • Identification of SERPINA12 variants in human disease (palmoplantar keratoderma) reveals unexpected connections between metabolism and skin disorders

Current research trajectories suggest Serpin A12 functions as part of an integrated response system to metabolic stress, with potential as both a biomarker and therapeutic target for obesity-related metabolic disorders.

What are emerging connections between Serpin A12 and unexpected pathophysiological processes?

Recent research has revealed unexpected connections between Serpin A12 and diverse pathophysiological processes beyond its established metabolic functions:

• Dermatological disorders:

  • Loss-of-function variants in human SERPINA12 have been implicated in autosomal recessive diffuse hereditary palmoplantar keratoderma (hPPK)

  • This reveals an unexpected role in skin barrier function and keratinocyte biology

  • The disorder shares similarities with Nagashima-type PPK caused by variants in another serpin, SERPINB7

  • European patients with a novel SERPINA12 c.1100G>A p.(Gly367Glu) missense variant demonstrate that the variant spectrum extends beyond previously reported Asian populations

• Bone metabolism:

  • Serpin A12 inhibits TRANCE/RANK L-induced osteoclast development

  • This suggests potential roles in bone density regulation and osteoporosis prevention

  • Creates potential connections between metabolic health and skeletal integrity

• Vascular biology:

  • Inhibitory effects on inflammatory activation of vascular smooth muscle and endothelial cells

  • Potential implications for vascular complications in metabolic disease

  • Suggests broader anti-inflammatory properties beyond adipose tissue

• Protease networks beyond KLK7:

  • The structural similarity between SERPINA12 and SERPINB7-related keratodermas suggests common mechanisms in protease regulation

  • Understanding these connections may reveal broader regulatory networks involving multiple serine proteases

These emerging connections highlight Serpin A12 as a multifunctional regulator involved in diverse physiological processes, suggesting potential therapeutic applications beyond metabolic disease and new directions for understanding tissue-specific protease regulation.

What are the most promising therapeutic applications of recombinant Serpin A12 research?

Recombinant Serpin A12 research presents several promising therapeutic applications based on its demonstrated biological activities:

• Metabolic disease interventions:

  • Treatment of obesity-related insulin resistance: Recombinant vaspin treatment counteracts obesity-induced inflammation and dysfunction of adipose tissue and liver and partly restores glucose tolerance

  • Prevention of metabolic deterioration: Transgenic models demonstrate resistance to obesogenic conditions, suggesting preventive potential

  • Liver steatosis protection: Effects on liver metabolism suggest applications in non-alcoholic fatty liver disease

• Novel delivery approaches:

  • Long-acting formulations to overcome potential pharmacokinetic limitations

  • Tissue-targeted delivery systems to enhance effects in specific metabolic tissues

  • Gene therapy approaches based on successful transgenic models showing metabolic benefits of sustained expression

• Combination therapies:

  • Synergistic potential with established diabetes treatments

  • Anti-inflammatory/insulin-sensitizing dual-action therapy

  • Adjunctive therapy for patients with suboptimal response to standard treatments

• Specialized applications based on emerging roles:

  • Dermatological applications: Investigation of topical applications for keratoderma based on SERPINA12 mutation associations

  • Bone metabolism: Potential applications in conditions with accelerated bone resorption

  • Vascular protection: Investigation for diabetic vascular complications

• Biomarker development:

  • Diagnostic markers for metabolic disease risk stratification

  • Predictive biomarkers for treatment response

  • Monitoring markers for disease progression

The most advanced therapeutic direction appears to be metabolic disease intervention, supported by multiple lines of evidence showing that both transgenic overexpression and recombinant protein administration improve metabolic parameters in obesity and insulin resistance models . The demonstration that extremely high circulating levels (>200 ng/ml) in transgenic mice enhance energy expenditure and limit diet-induced obesity provides strong preclinical rationale for therapeutic development .