Recombinant Human Fibroblast growth factor 21 protein (FGF21) (Active)

What is FGF21 and what are its primary biological functions?

FGF21 is a member of the FGF19 subfamily with hormone-like functions that predominantly originates from the liver. Unlike other FGF subfamilies, FGF21 requires βKlotho as a co-factor to increase its binding affinity to FGF receptors . FGF21 plays crucial roles in glucose and lipid metabolism by:

• Regulating glucose uptake in adipocytes

• Enhancing fatty acid oxidation

• Improving insulin sensitivity

• Increasing energy expenditure

• Regulating mitochondrial function and biogenesis

FGF21 functions through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), resulting in enhanced mitochondrial oxidative function . This activation cascade leads to increased expression of genes involved in mitochondrial function, including carnitine palmitoyltransferase 1A (CPT1a), isocitrate dehydrogenase 3 alpha (Idh3a), and cytochrome c (CytC) .

How do physiological and lifestyle factors influence endogenous FGF21 levels?

Multiple factors affect serum FGF21 levels, which researchers should consider when designing experiments or interpreting results:

Multivariable regression analysis has identified age, ALT, γ-GTP, smoking status, and breakfast and alcohol consumption frequency as independent variables for FGF21 levels . Additionally, the relationships between FGF21 and certain parameters (WC, SBP, TC) appear to differ between obese and non-obese individuals, with correlations observed only in non-obese subjects .

What experimental models are commonly used to study FGF21 function?

Several experimental models have proven valuable for FGF21 research:

• In vitro models:

  • 3T3-L1 adipocytes for studying adipose tissue effects

  • Primary human adipocytes for translational relevance

  • Cardiomyocyte cultures for cardiac effects

• In vivo models:

  • FGF21 knockout mice (FGF21KO) to study loss-of-function

  • Adipose-specific βklotho knockout mice to investigate tissue-specific effects

  • Diet-induced obesity models (high-fat diet)

  • Genetic models of obesity (db/db mice)

  • Specific disease models:

    • Uremic cardiomyopathy rats

    • Hypertension mice

    • Angiotensin II-induced cardiac hypertrophy mice

These models have revealed that FGF21 increases mitochondrial gene expression and protein levels in adipocytes , protects against cardiac hypertrophy , and improves insulin sensitivity by promoting subcutaneous adipose tissue expansion .

What are the optimal methods for producing and purifying recombinant human FGF21 protein?

Production of high-quality recombinant human FGF21 requires attention to several methodological considerations:

• Expression Systems:

  • E. coli systems are most commonly used due to high yield and cost-effectiveness

  • Mammalian expression systems (CHO cells) may provide better post-translational modifications

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Size exclusion chromatography to remove aggregates and ensure monodispersity

  • Anion exchange chromatography for final polishing

• Quality Control Considerations:

  • Confirmation of correct folding using circular dichroism

  • Verification of biological activity through cell-based assays (glucose uptake in adipocytes)

  • Endotoxin removal and testing (critical for in vivo experiments)

• Stability Enhancement:

  • Addition of stabilizing excipients (e.g., trehalose)

  • Optimization of buffer conditions (pH 7.0-7.5 typically optimal)

  • Storage at -80°C with minimized freeze-thaw cycles

For research requiring enhanced pharmaceutical properties, strategies include extending FGF21's half-life and enhancing its affinity to β-Klotho through targeted mutagenesis and screening .

How should researchers measure and interpret FGF21 resistance in experimental and clinical settings?

FGF21 resistance is a complex phenomenon observed in several metabolic conditions where elevated FGF21 levels fail to elicit the expected physiological responses. Key methodological approaches include:

• Assessment Methods:

  • Measuring the ratio of FGF21 to adiponectin (FGF21/adiponectin ratio can predict glycemic deterioration)

  • Evaluating downstream signaling (phosphorylation of ERK1/2 in target tissues)

  • βKlotho expression analysis in target tissues

  • Functional tests (glucose tolerance, insulin sensitivity) with FGF21 administration

• Interpretation Challenges:

  • High baseline FGF21 levels in T2DM patients suggest resistance

  • Paradoxical findings: FGF21 levels are higher in diabetic patients despite its anti-diabetic effects

  • The prognostic value varies by context (e.g., in diabetes patients with coronary artery calcification, lower FGF21 levels predict better long-term prognosis)

• Experimental Controls:

  • Include adipose-specific βklotho knockout models to confirm FGF21 dependency

  • Use tissue-specific deletion models to identify sites of resistance

  • Employ dose-response studies to characterize the resistance phenotype

Understanding FGF21 resistance is crucial as T2DM patients show significantly higher serum FGF21 levels compared to control groups , suggesting potential adaptation mechanisms.

What approaches can be used to enhance FGF21 efficacy for experimental applications?

Researchers have developed several strategies to overcome FGF21's inherent limitations:

• Structural Modifications:

  • Site-directed mutagenesis to enhance β-Klotho binding affinity

  • N-terminal modifications to reduce proteolytic degradation

  • PEGylation to extend half-life

• Fusion Proteins:

  • Fc-fusion proteins for extended circulation

  • GLP-1-Fc-FGF21 dual agonists that exhibit potent and sustained effects on glucose lowering, weight loss, and lipid profile improvement

  • Creation of chimeric proteins with complementary metabolic activities

• Delivery Systems:

  • Encapsulation in nanoparticles for targeted delivery

  • Gene therapy approaches for sustained endogenous production

  • Tissue-specific targeting strategies

• Combination Therapies:

  • Co-administration with other metabolic regulators (e.g., adiponectin inducers)

  • Use with AMPK or SIRT1 activators to enhance downstream signaling

These approaches address the poor pharmacokinetics and biophysical properties of native FGF21, expanding its experimental utility and therapeutic potential .

How does FGF21 signaling differ among target tissues?

FGF21 exhibits tissue-specific effects through differential receptor expression and downstream signaling:

• Adipose Tissue:

  • High expression of βKlotho and FGF receptors

  • Primary site for insulin-sensitizing effects

  • Upregulates adiponectin secretion, particularly in subcutaneous adipose tissue (SAT)

  • Promotes "browning" of white adipose tissue through PGC-1α induction

  • SAT is essential for FGF21-mediated systemic insulin sensitivity improvement

• Liver:

  • Major site of FGF21 production

  • FGF21 inhibits liver mTORC1 to increase insulin sensitivity

  • Hepatic insulin resistance is exacerbated in FGF21-deficient mice

• Heart:

  • FGF21 protects against cardiac hypertrophy by regulating SIRT1 deacetylase activity

  • Prevents cardiac fibrosis in hypertensive heart disease models

  • Attenuates cardiac derangements by regulating autophagy in cardiomyocytes

• Central Nervous System:

  • Influences energy expenditure and food intake

  • Requires βKlotho expression in specific hypothalamic nuclei

Understanding these tissue-specific effects is crucial for experimental design, as tissue-specific knockout models reveal distinct phenotypes.

What molecular mechanisms underlie FGF21's beneficial metabolic effects?

FGF21 activates multiple signaling cascades that collectively improve metabolic homeostasis:

• AMPK-SIRT1-PGC-1α Axis:

  • FGF21 increases AMPK phosphorylation in adipocytes

  • Activated AMPK promotes SIRT1 activity

  • SIRT1 deacetylates PGC-1α, enhancing its transcriptional activity

  • This cascade increases mitochondrial biogenesis and function

• Adiponectin Pathway:

  • FGF21 upregulates adiponectin in subcutaneous adipose tissue

  • Adiponectin mediates many of FGF21's insulin-sensitizing effects

  • FGF21 inhibits endoplasmic reticulum stress in adipose tissue to release suppression of adiponectin expression

• Anti-inflammatory Effects:

  • Reduces inflammatory markers in serum and white adipose tissue

  • Inhibits NF-κB signaling in adipocytes

  • Increases M2 macrophage polarization in adipose tissue

• Autophagy Regulation:

  • FGF21 regulates autophagy in cardiomyocytes and other tissues

  • FGF21-deficient mice show impaired autophagy leading to lipid accumulation and cardiac derangements

These interconnected pathways explain how FGF21 simultaneously improves glucose homeostasis, increases energy expenditure, and protects against lipotoxicity.

How does FGF21 interact with other metabolic hormones and regulatory pathways?

FGF21 functions within a complex network of metabolic regulators:

• Interactions with Insulin Signaling:

  • FGF21 increases insulin sensitivity through multiple mechanisms

  • Can partially restore insulin signaling even in insulin-resistant states

  • Reduces hepatic glucose production and enhances peripheral glucose uptake

• Cross-talk with Glucagon and GLP-1:

  • Synergistic effects with GLP-1 have led to the development of dual agonists

  • Counters some glucagon effects while reinforcing others (e.g., both increase energy expenditure)

• Adipokine Network:

  • FGF21 increases adiponectin production

  • The FGF21/adiponectin ratio predicts glycemic progression

  • May influence leptin sensitivity in the central nervous system

• Circadian Rhythms and Fasting Response:

  • FGF21 expression shows circadian variation

  • Acts as a stress-responsive hormone under various metabolic stresses

  • Mediates many of the metabolic adaptations to fasting and ketogenic diets

Understanding these interactions helps explain the context-dependent effects of FGF21 and informs experimental design for metabolic studies.

How can FGF21 be used as a biomarker in metabolic and cardiovascular diseases?

FGF21 shows promise as a biomarker in several conditions:

• Type 2 Diabetes:

  • Higher FGF21 levels indicate increased risk of glycemic progression over five years

  • FGF21/adiponectin ratio predicts deterioration in glycemia

  • Serum FGF21 appears to be an alternative to glucose tolerance testing for diabetes prediction

  • Lower urinary glucose excretion in T2DM correlates with higher serum FGF21

• Cardiovascular Disease:

  • Elevated in heart failure with reduced ejection fraction (HFrEF)

  • Potential diagnostic marker for heart failure with preserved ejection fraction (HFpEF)

  • Reflects diastolic dysfunction

  • In diabetes patients with coronary artery calcification, lower FGF21 levels predict better long-term prognosis

• Obesity and NAFLD:

  • Elevated in obesity, correlating with multiple metabolic parameters

  • May indicate hepatic stress in non-alcoholic fatty liver disease

• Methodological Considerations:

  • Standardized collection protocols are essential (time of day, fasting status)

  • Account for confounding factors (age, BMI, liver function, lifestyle)

  • Paired assessment with other markers (adiponectin, inflammatory markers) provides context

The paradoxical increase of FGF21 in metabolic diseases suggests it serves as a defense mechanism to protect against systemic insulin resistance .

What are the key experimental design considerations for FGF21 research in disease models?

Researchers should consider several factors when designing FGF21 studies:

• Model Selection:

  • Choose models that recapitulate human pathophysiology

  • Consider tissue-specific knockout models to isolate mechanisms

  • FGF21KO mice show less subcutaneous adipose tissue mass and greater insulin resistance on high-fat diet

  • Adipose-specific βklotho knockout mice help identify FGF21-dependent effects

  • Transplantation of subcutaneous adipose tissue from wild-type to FGF21KO mice improves insulin sensitivity

• Dosing and Administration:

  • Pharmacokinetic considerations (native FGF21 has a short half-life)

  • Route of administration affects tissue distribution

  • Consider replenishment to physiological vs. pharmacological levels

  • Native FGF21 vs. modified versions with enhanced properties

• Outcome Measures:

  • Comprehensive metabolic phenotyping (glucose tolerance, insulin sensitivity)

  • Tissue-specific molecular readouts (gene expression, protein phosphorylation)

  • Histological assessment of target tissues

  • Functional outcomes relevant to the disease model

• Controls and Comparators:

  • Paired-feeding controls to account for food intake effects

  • Comparison with established therapies

  • Dose-ranging studies to establish dose-response relationships

Thorough experimental design is essential as FGF21's effects are context-dependent and involve multiple mechanisms.

What are the emerging therapeutic applications of engineered FGF21 variants?

FGF21-based therapeutics show promise for multiple conditions:

• Type 2 Diabetes and Obesity:

  • FGF21 analogs improve dyslipidemia and decrease body weight in patients with obesity and T2DM

  • Reduce fasting insulin levels and increase adiponectin levels

  • GLP-1-Fc-FGF21 dual agonists show potent effects on glucose lowering, weight loss, and lipid profile improvement

• Non-alcoholic Steatohepatitis (NASH):

  • FGF21 reduces hepatic steatosis, inflammation, and fibrosis

  • GLP-1-Fc-FGF21 dual agonists show promise for NASH treatment

• Cardiovascular Protection:

  • Potential to prevent cardiac remodeling and heart failure

  • Protection against cardiac hypertrophy in pathological models

  • Attenuation of cardiac fibrosis

• Engineering Approaches:

  • Half-life extension through mutagenesis

  • Enhanced β-Klotho affinity through targeted modifications

  • Creation of dual-targeted agonists (e.g., GLP-1-Fc-FGF21)

  • Development of small molecule FGF21 mimetics or receptor activators

Recent clinical development has focused on overcoming FGF21's poor pharmacokinetics and biophysical properties through novel engineering approaches .

What are the unresolved questions regarding FGF21 biology and function?

Several knowledge gaps remain in FGF21 research:

• Tissue-Specific Production and Action:

  • Relative contribution of FGF21 from different tissues under various conditions

  • Complete mapping of tissue-specific receptor expression and signaling

  • Role of autocrine/paracrine vs. endocrine FGF21 actions

• Resistance Mechanisms:

  • Molecular basis of FGF21 resistance in metabolic diseases

  • Relationship between chronic elevation and diminished response

  • Strategies to overcome resistance

• Central vs. Peripheral Effects:

  • Neural circuits mediating central FGF21 effects

  • Blood-brain barrier transport mechanisms

  • Integration of central and peripheral signals

• Circadian and Environmental Regulation:

  • Complete understanding of circadian FGF21 production and action

  • Impact of environmental factors (temperature, stress, microbiome)

  • Nutritional regulation beyond fasting and ketogenic states

Research addressing these questions will provide a more comprehensive understanding of FGF21 biology and guide therapeutic development.

What novel methodologies are advancing FGF21 research?

Cutting-edge approaches are transforming FGF21 research:

• Single-Cell Technologies:

  • Single-cell RNA sequencing to identify responding cell populations

  • Mass cytometry for comprehensive signaling analysis

  • Spatial transcriptomics to map tissue responses

• Advanced Genetic Models:

  • Inducible and tissue-specific knockout/knockin systems

  • CRISPR-based screening for FGF21 modifiers

  • Humanized mouse models expressing human FGF21 and receptors

• Computational Approaches:

  • Systems biology modeling of FGF21 networks

  • AI-driven prediction of optimal FGF21 variants

  • Virtual screening for small molecule modulators

• Imaging Technologies:

  • PET imaging with labeled FGF21 to track tissue distribution

  • Functional MRI to assess metabolic effects in real-time

  • Intravital microscopy to visualize cellular responses

These methodologies promise to accelerate understanding of FGF21 biology and facilitate therapeutic development.

How might FGF21 research inform broader metabolic regulatory networks?

FGF21 research offers insights into fundamental metabolic principles:

• Integrative Physiology:

  • FGF21 exemplifies cross-tissue communication in metabolic regulation

  • Illustrates how the body adapts to nutritional and environmental challenges

  • Demonstrates the concept of beneficial stress responses (hormesis)

• Evolutionary Biology:

  • FGF21's role in adaptation to fasting and cold exposure

  • Comparative analysis across species reveals evolutionary conservation

  • Potential role in human evolutionary adaptation to dietary patterns

• Systems Approaches to Metabolism:

  • FGF21 as a node in complex metabolic networks

  • Interaction with multiple regulatory systems (insulin/glucagon, sympathetic nervous system)

  • Example of how single factors can coordinate diverse metabolic processes

• Therapeutic Paradigms:

  • Model for developing multi-functional therapeutic proteins

  • Engineering strategies applicable to other biological therapeutics

  • Concept of targeting metabolic pathways at multiple points simultaneously

FGF21 research thus contributes to our broader understanding of metabolic regulation and informs approaches to other metabolic diseases.