FGF 21 Mouse, Sf9

What is the molecular structure of mouse FGF-21 produced in Sf9 cells?

Mouse FGF-21 produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 190 amino acids (residues 29-210) with a molecular mass of 21.0 kDa. On SDS-PAGE, the protein typically appears at approximately 18-28 kDa due to its glycosylation pattern. Commercial preparations generally include an 8 amino acid His tag at the C-terminus to facilitate purification processes . The protein belongs to the fibroblast growth factor family, which comprises more than 20 small secreted peptides initially characterized for their ability to stimulate fibroblast proliferation. Mouse FGF-21 shares 81% amino acid sequence identity with human FGF-21, 80% with bovine, 79% with canine, and 92% with rat FGF-21 .

How is recombinant mouse FGF-21 typically formulated for research applications?

Standard research-grade mouse FGF-21 protein solution is typically supplied at a concentration of 0.5 mg/ml in Phosphate Buffered Saline (pH 7.4) containing 10% glycerol as a stabilizer . The addition of glycerol helps maintain protein stability during freeze-thaw cycles. Purity levels greater than 90.0% as determined by SDS-PAGE are standard for most research applications. When working with this protein, it's important to note that it is intended for research use only and not for therapeutic applications or human/animal diagnostic use .

What are the primary metabolic functions of FGF-21 in mouse models?

Mouse FGF-21 functions as a critical metabolic regulator with multiple effects on energy homeostasis. Studies have demonstrated that FGF-21 promotes glucose uptake in adipocytes, improves insulin sensitivity, stimulates ketogenesis during fasting states, and enhances fat utilization . In diet-induced obese mice and ob/ob mice, systemic administration of FGF-21 for 2 weeks resulted in approximately 20% reduction in body weight, primarily through decreased adiposity. FGF-21-treated animals exhibited increased energy expenditure, enhanced fat utilization, improved lipid excretion, reduced hepatosteatosis, and ameliorated hyperglycemia . Additionally, transgenic mice overexpressing FGF-21 showed protection against diet-induced obesity, suggesting its potential as a metabolic regulator with therapeutic implications .

How does FGF-21 influence thermogenesis and energy expenditure in mice?

FGF-21 plays a significant role in regulating thermogenesis and energy metabolism through its actions on adipose tissue. Recent studies have established that brown adipose tissue is not only a target for FGF-21 but also potentially an important source of systemic FGF-21, suggesting an autocrine-paracrine axis that regulates thermogenic functions . In obese mice with ad libitum access to food, continuous infusion of FGF-21 (1 mg/kg/day) for 5 days resulted in a small but significant elevation of core body temperature 4 hours after treatment initiation, lasting approximately 10 hours. FGF-21 administration also increased energy expenditure during both light and dark cycles and depressed the respiratory quotient during dark cycles, indicating enhanced fat utilization . Interestingly, the effects of FGF-21 on core temperature appear to be dependent on nutritional status, with divergent effects observed in fed versus fasting states.

What methods are most effective for detecting mouse FGF-21 in experimental samples?

Several validated methods exist for detecting and quantifying mouse FGF-21 in research settings:

Western Blot Analysis: Mouse FGF-21 can be detected using specific antibodies, such as Goat Anti-Mouse FGF-21 Antigen Affinity-purified Polyclonal Antibody. When analyzed under reducing conditions using immunoblot buffer group 1, FGF-21 appears as a specific band at approximately 26 kDa. For optimal results, PVDF membranes probed with 0.25 μg/mL of antibody followed by HRP-conjugated secondary antibody have shown good specificity in detecting FGF-21 in tissue lysates including mouse and rat spleen tissues .

Quantitative RT-PCR: For gene expression analysis, quantitative RT-PCR can effectively measure FGF-21 mRNA levels in various tissues, particularly in muscle samples where FGF-21 expression correlates with metabolic status .

Plasma Concentration Measurement: ELISA-based methods can detect circulating FGF-21 levels in plasma samples from experimental animals, allowing for correlation between systemic FGF-21 levels and metabolic parameters .

What experimental considerations are important when studying the effects of FGF-21 in mouse models?

When designing experiments to investigate FGF-21 function in mice, several critical factors should be considered:

Nutritional Status: The metabolic effects of FGF-21 can vary significantly depending on whether animals are in fed or fasted states. For instance, lower core body temperatures were observed in lean, starved FGF-21 transgenic mice but not in fed animals .

Duration of Treatment: Acute versus chronic administration of FGF-21 may produce different metabolic outcomes. Short-term studies (5 days) have shown changes in core temperature and energy expenditure, while longer treatments (2 weeks) demonstrated significant effects on body weight and adiposity .

Dosage: Typical effective doses in mouse studies range from 0.5-1 mg/kg/day, often administered via continuous infusion or daily injection protocols .

Control Groups: Appropriate controls should include wild-type littermates for transgenic models or vehicle-treated animals for exogenous FGF-21 administration studies to account for strain-specific responses .

How does mouse FGF-21 differ from human FGF-21, and why is this important for translational research?

A critical distinction between mouse and human FGF-21 exists in their susceptibility to proteolytic processing. Human FGF-21 has a limited half-life in circulation due to proteolytic cleavage between P171 and S172 by fibroblast activation protein (FAP). In contrast, mouse FGF-21 lacks this specific FAP cleavage site and is therefore not cleaved by FAP . This fundamental difference has significant implications for translational research and the development of FGF-21-based therapeutics.

The differential susceptibility to FAP cleavage means that pharmacokinetic and pharmacodynamic studies in mice may not accurately predict human responses. Specifically, interventions targeting FAP to extend FGF-21 half-life would be effective in humans but show no effect in mice. This species-specific difference creates challenges for pre-clinical proof-of-concept studies in rodent models when developing FAP inhibitors as potential adjunctive therapies for FGF-21-based treatments of metabolic disorders .

What receptor systems mediate FGF-21 signaling, and do they differ between mice and humans?

FGF-21 signals through FGF receptors (FGFRs), with at least five distinct subfamilies involved: FGFR-1, -2, -3, and -4, all possessing intrinsic tyrosine kinase activity, and FGFR-5, which lacks an intracellular kinase domain . These receptors play important roles in regulating glucose and lipid homeostasis across species.

Research has demonstrated that proper FGF signaling is crucial for normal β-cell function and glycemia maintenance, as evidenced by the development of diabetes in mice overexpressing a dominant negative form of FGFR-1 in β cells. FGFR-2 appears to be a key mediator during pancreatic development, while FGFR-4 has been implicated in cholesterol metabolism and bile acid synthesis . While the basic receptor machinery is conserved between mice and humans, differences in tissue-specific expression patterns and downstream signaling pathways may contribute to species-specific responses to FGF-21 treatment.

How does FGF-21 interact with cellular stress response pathways?

FGF-21 expression and function appear to be closely linked with cellular stress response mechanisms, particularly in muscle tissue. Gene expression analysis and protein studies have revealed that FGF-21 induction correlates with activation of integrated stress response pathways . In transgenic mouse models, Western blot analysis has demonstrated associations between FGF-21 levels and stress response markers such as HSP25 and HSP70 in quadriceps muscle .

The relationship between AMPK signaling and FGF-21 expression suggests FGF-21 may serve as part of an energy-sensing network that responds to metabolic stress. Studies of double transgenic mice expressing both dominant-negative AMPK α2 and UCP1 show altered FGF-21 expression patterns compared to wild-type controls, with concurrent changes in cellular stress response markers . These findings suggest FGF-21 may function as both a responder to and mediator of cellular stress adaptation pathways, particularly in metabolically active tissues.

What methodological approaches are recommended for studying FGF-21's role in adipose tissue "browning"?

To effectively investigate FGF-21's role in promoting the conversion of white adipose tissue to brown adipose tissue (browning), researchers should consider the following methodological approaches:

Histological Analysis: Examination of adipose tissue morphology after FGF-21 treatment, looking for multilocular lipid droplets characteristic of brown/beige adipocytes.

Molecular Marker Assessment: Quantification of brown adipose tissue-specific markers such as UCP1, PRDM16, and PGC-1α at both mRNA and protein levels following FGF-21 administration.

Mitochondrial Function Analysis: Measurement of mitochondrial content, respiratory capacity, and thermogenic potential in adipose tissues from FGF-21-treated animals.

In Vivo Metabolic Phenotyping: Comprehensive assessment of energy expenditure, core temperature, and cold tolerance in response to FGF-21 treatment under various thermal challenges .

Ex Vivo Adipocyte Studies: Isolation and culture of primary adipocytes to directly assess FGF-21's effects on cellular browning processes and thermogenic gene expression programs.

What are the optimal storage and handling conditions for maintaining FGF-21 activity?

To preserve the biological activity of recombinant mouse FGF-21, researchers should store the protein solution at -80°C for long-term storage or at -20°C for shorter periods. The inclusion of 10% glycerol in the formulation helps maintain stability during freeze-thaw cycles . Aliquoting the stock solution is recommended to minimize repeated freeze-thaw cycles, which can compromise protein integrity. When working with the protein, it should be thawed gently on ice and handled using low-protein binding tubes and pipette tips to prevent loss through adsorption.

For experiments requiring extended incubation periods, stability tests at the experimental temperature should be conducted to ensure the protein maintains activity throughout the study duration. The use of protease inhibitors may be warranted in certain experimental systems, even though mouse FGF-21 is not susceptible to FAP cleavage, as other proteases may affect its stability .

How can researchers verify the biological activity of recombinant mouse FGF-21 preparations?

Before conducting complex metabolic studies, validating the biological activity of recombinant mouse FGF-21 is crucial. Several approaches can be employed:

• Adipocyte Glucose Uptake Assay: FGF-21 is a potent activator of glucose uptake in adipocytes, making this a functional test of bioactivity .

• Receptor Phosphorylation Assay: Measuring the phosphorylation of FGF receptors and downstream signaling molecules (ERK1/2, Akt) in responsive cell lines following FGF-21 treatment.

• Gene Expression Analysis: Quantifying the induction of FGF-21-responsive genes in appropriate cell types or tissues.

• Pilot In Vivo Studies: Small-scale pilot studies measuring established FGF-21 effects, such as changes in blood glucose levels or energy expenditure, can confirm in vivo bioactivity before larger experiments.

• Thermal Shift Assay: This technique can assess protein folding and stability, which correlate with biological function.

What are promising research areas involving mouse FGF-21 for metabolic disease modeling?

Several cutting-edge research directions offer significant potential for advancing our understanding of FGF-21 biology and its therapeutic applications:

Tissue-Specific FGF-21 Signaling: Investigating the differential effects of FGF-21 across tissues using conditional knockout or tissue-specific overexpression models could reveal new metabolic regulatory mechanisms .

Circadian Regulation: Exploring FGF-21's role in circadian metabolic rhythms, particularly how it affects energy expenditure differently during light and dark cycles .

Inter-Organ Communication: Studying how FGF-21 mediates communication between liver, adipose tissue, muscle, and other metabolic organs could uncover new endocrine networks .

Stress-Induced Metabolic Adaptation: Further characterization of how FGF-21 links cellular stress responses to systemic metabolic adaptation may reveal novel therapeutic targets .

Engineered FGF-21 Variants: Developing mouse models expressing modified FGF-21 proteins with enhanced stability or receptor specificity could advance translational research aimed at therapeutic applications .

How might differences between mouse and human FGF-21 be addressed in translational research?

To overcome the challenges posed by species-specific differences in FGF-21 biology, researchers might consider several innovative approaches:

Humanized Mouse Models: Generating mice that express human FGF-21 rather than the mouse ortholog would provide a more relevant system for testing human-focused interventions, particularly those targeting FAP-mediated degradation .

Parallel Testing Platforms: Simultaneously testing compounds in both mouse and human systems (cell lines, primary tissues, etc.) could help identify species-specific responses early in the research process.

Structural Modifications: Engineering mouse FGF-21 to include human-specific features (such as the FAP cleavage site) might create better translational models for specific research questions.

Comparative Pharmacology: Detailed side-by-side analysis of pharmacokinetic and pharmacodynamic properties of mouse and human FGF-21 could help develop mathematical models to better predict human responses from mouse data.

Alternative Animal Models: For certain aspects of FGF-21 biology, particularly those related to proteolytic processing, models other than mice (such as monkeys, which share the FAP cleavage site with humans) might be more appropriate for translational studies .