Visfatin Human, His

What is human visfatin and what is its genomic location?

Human visfatin is an adipokine and enzyme initially identified as Pre-B cell colony-enhancing factor (PBEF) expressed in lymphocytes. The human visfatin gene is located on chromosome 7 (7q22.1 to 7q31.33) and encodes a protein comprised of 491 amino acids with a molecular weight of 52 kDa . The gene has been effectively maintained throughout evolution, suggesting important biological functions . Visfatin exhibits insulin-mimetic effects, binding to and activating insulin receptors, thus potentially playing a role in glucose homeostasis .

What is the significance of histidine-tagged visfatin in research applications?

Histidine-tagged (His-tagged) human visfatin refers to recombinant visfatin protein produced with a polyhistidine tag, typically added to the N or C-terminus. This modification enables efficient purification using immobilized metal affinity chromatography with minimal impact on protein structure or function. For research applications, His-tagged visfatin allows for:

• High-purity protein isolation for in vitro studies

• Consistent protein preparation across experiments

• Antibody detection using anti-His antibodies as an alternative to visfatin-specific antibodies

• Immobilization on surfaces for protein interaction studies

When using His-tagged visfatin, researchers should validate that the tag does not interfere with the specific biological activity being studied.

What are the primary physiological functions of visfatin?

Visfatin demonstrates multiple physiological functions with significant implications for metabolism and inflammation:

• Enzymatic role as NAMPT in NAD biosynthesis pathway

• Insulin-mimetic effects through insulin receptor binding and activation

• Regulation of steroidogenesis in human granulosa cells

• Enhancement of RANKL-induced osteoclastogenesis affecting bone metabolism

• Immunomodulatory functions in various inflammatory conditions

• Potential role in glucose homeostasis evidenced by elevated plasma glucose in visfatin gene heterozygous mutant mice

What are the optimal methods for measuring visfatin in biological samples?

Multiple validated approaches exist for visfatin detection and quantification:

ELISA-Based Methods:

• Competition-based ELISA offers high sensitivity (0.778 ng/ml) with a detection range of 0.1-1,000 ng/ml

• Recommended dilution for human serum/plasma is typically 4X

• Shows specificity with no cross-reactivity with other cytokines including Ghrelin, Nesfatin, NPY and APC

Genetic Analysis:

• RT-PCR for mRNA expression analysis using specific primers designed with Primer-1® software and verified by BLAST®

• Genotyping of visfatin SNPs (rs2302559 and rs1215113036) can be performed using tetra-primer amplification refractory mutation system (tARMS) PCR

• DNA sequencing provides confirmation of genetic variants using analysis software like Mega11

Immunodetection Methods:

• Immunohistochemistry has successfully identified visfatin in human follicles, granulosa cells, cumulus cells, and oocytes

• Western blotting can determine protein expression levels in tissue homogenates and cell lysates

How should experimental designs account for visfatin's effects on the RANKL pathway?

When investigating visfatin's role in RANKL-induced osteoclastogenesis, researchers should consider:

Experimental Design Considerations:

• Although visfatin alone shows modest osteoclast-inductive effects, co-stimulation with RANKL significantly enhances osteoclast differentiation and activation

• Neutralization experiments using visfatin-blocking antibodies demonstrate near-complete suppression of RANKL-induced osteoclastogenesis

Key Molecular Readouts:

• Tartrate-resistant acid phosphatase-positive multinucleated osteoclast formation

• Expression levels of nuclear factor of activated T cells cytoplasmic 1

• Activation of nuclear factor-κB and mitogen-activated protein kinase signaling pathways

• Quantification of resorption pit formation as functional outcome measure

Recommended Controls:

• RANKL-only stimulation

• Visfatin-only stimulation

• Combined RANKL and visfatin with varying concentrations

• Isotype control antibodies for neutralization experiments

What methodological approaches are recommended for studying visfatin regulation by insulin sensitizers?

Studies examining visfatin regulation by insulin sensitizers should incorporate:

Treatment Conditions:

• Metformin dose-response (0.1, 1, and 10 mM) shows increased visfatin mRNA in a dose-dependent manner after 24h

• Rosiglitazone increases visfatin mRNA expression only at higher concentrations (10 μM) after 24h

• Both compounds reduce visfatin expression after 48h, indicating time-dependent effects

Signaling Pathway Analysis:

• AMPK/SIRT1 signaling pathways mediate metformin effects on visfatin expression

• Include Compound C (AMPK inhibitor) and Aicar (AMPK activator) experiments

• Incorporate Sirtinol (SIRT1 inhibitor) to assess sirtuin involvement

Cell Models:

• Primary human granulosa cells provide physiologically relevant context

• KGN human ovarian granulosa-like tumor cell line offers reproducible alternative

• Consider comparing adipose tissue-derived cells with other visfatin-expressing cell types

Which visfatin gene polymorphisms show significant association with metabolic syndrome?

Research has identified two key visfatin SNPs with strong metabolic syndrome associations:

Both SNPs show significantly higher mutant allele frequencies in metabolic syndrome patients compared to controls . The heterozygous mutant genotype (GA) for rs1215113036 occurs 129-fold more frequently in metabolic syndrome patients . These associations suggest visfatin genetic variants play a significant role in metabolic syndrome pathophysiology.

How does visfatin expression differ between visceral and subcutaneous adipose tissue?

Visfatin shows differential expression between adipose tissue depots:

• Preferentially expressed in visceral adipose tissue compared to subcutaneous adipose tissue

• Expression levels in plasma increase during obesity development

• This tissue-specific expression pattern may explain the stronger association between visceral adiposity and metabolic disorders

Researchers should consider these depot-specific differences when designing experiments and interpreting results from adipose tissue samples.

What is the relationship between visfatin and insulin sensitivity in humans?

The relationship between visfatin and insulin sensitivity is complex:

• Visfatin binds to and activates the insulin receptor, exhibiting insulin-mimetic effects

• Mice heterozygous for visfatin gene mutation show modestly higher plasma glucose levels compared to wild-type littermates

• Visfatin exerts insulin-mimetic effects in cultured cells and can lower plasma glucose levels in mice

• Visfatin enhances IGF-1-induced (but not FSH-induced) steroidogenesis and cell proliferation

• Rapidly activates mitogen-activated protein kinase pathways via ERK1/2, P38, and Akt phosphorylation

These findings suggest visfatin may function as an insulin-sensitizing factor under certain physiological conditions.

How can researchers differentiate between visfatin's enzymatic and cytokine functions?

Distinguishing visfatin's dual roles requires specific methodological approaches:

Enzymatic Function (NAMPT) Assessment:

• Measure NAD biosynthesis activity using enzymatic assays

• Employ specific NAMPT inhibitors (e.g., FK866) to selectively block enzymatic function

• Monitor changes in cellular NAD+/NADH ratios

• Use site-directed mutagenesis to disrupt catalytic sites while preserving protein structure

Cytokine Function Assessment:

• Evaluate receptor binding and activation (particularly insulin receptor)

• Assess downstream signal transduction pathways

• Measure physiological outcomes like glucose uptake or steroidogenesis

• Use neutralizing antibodies that block receptor interaction but not enzymatic activity

Experimental Strategies:

• Compare wild-type visfatin with catalytically inactive mutants

• Utilize domain-specific blocking antibodies

• Perform structure-function analysis with truncated proteins

• Employ subcellular fractionation to separate intracellular vs. secreted visfatin

What are the critical considerations for studying visfatin in reproductive contexts?

Research on visfatin's reproductive roles should address:

Tissue Expression Patterns:

• Visfatin is expressed in human granulosa cells, cumulus cells, and oocytes

• Expression patterns may vary throughout follicular development and menstrual cycle

• Consider potential paracrine/autocrine signaling within follicular microenvironment

Functional Assays:

• Visfatin (10 ng/ml) significantly increases IGF-1-induced (but not FSH-induced) progesterone and estradiol secretion

• Enhances IGF-1-induced thymidine incorporation in human granulosa and KGN cells

• Activates MAPK pathways via ERK1/2, P38, and Akt phosphorylation under basal conditions

Regulatory Mechanisms:

• Metformin and rosiglitazone differentially regulate visfatin expression in granulosa cells

• AMPK/SIRT1 signaling pathways mediate insulin sensitizer effects on visfatin expression

• Consider potential reproductive hormone feedback on visfatin production

How should researchers interpret conflicting data on visfatin's physiological roles?

When addressing contradictory findings, consider:

Experimental Variables:

• Source and purity of recombinant visfatin (bacterial vs. mammalian expression)

• Presence/absence and position of histidine tags

• Protein conformation and potential aggregation

• Concentration ranges used (physiological vs. pharmacological)

• Acute vs. chronic exposure paradigms

Model System Differences:

• In vitro vs. in vivo findings

• Species differences (human vs. rodent systems)

• Primary cells vs. cell lines

• Metabolic status of experimental models (normal vs. insulin resistant)

Methodological Approaches to Resolve Discrepancies:

• Perform side-by-side comparisons with standardized protocols

• Include appropriate positive and negative controls

• Use multiple complementary techniques to verify findings

• Validate in vitro observations in appropriate in vivo models

• Consider context-dependency of visfatin functions

What is the potential role of visfatin in bone metabolism disorders?

Emerging evidence links visfatin to bone metabolism through several mechanisms:

• Enhances RANKL-induced osteoclast differentiation from precursor cells

• Co-stimulation with RANKL significantly enhances osteoclast differentiation and activation

• Neutralizing visfatin with blocking antibodies suppresses RANKL-induced osteoclastogenesis

• Affects nuclear factor of activated T cells cytoplasmic 1 and osteoclast-specific proteins

• Influences nuclear factor-κB and mitogen-activated protein kinase signaling pathways

• Impacts resorption pit formation, indicating functional effects on bone resorption

Analysis of GEO datasets (GSE230665) shows altered visfatin expression in osteoporosis patients, suggesting clinical relevance . The RANKL/visfatin signaling axis represents a potential therapeutic target for bone loss-related diseases.

How might visfatin's dual functions influence experimental outcomes?

Visfatin's multifunctionality creates specific experimental challenges:

Dual Function Considerations:

• Enzymatic role as NAMPT in NAD biosynthesis

• Cytokine-like effects through receptor binding and activation

• Intracellular vs. extracellular actions

• Direct vs. indirect effects on target cells

Experimental Design Recommendations:

• Use specific inhibitors to distinguish enzymatic from cytokine functions

• Employ domain-specific mutations to selectively disrupt individual functions

• Consider compartmentalization (intracellular vs. secreted visfatin)

• Include appropriate controls for each functional aspect

• Assess both immediate signaling and long-term metabolic effects

Potential Confounding Factors:

• Endogenous visfatin production by experimental cell types

• NAD+ depletion effects independent of receptor activation

• Presence of visfatin-binding proteins in serum-containing media

• Cross-talk between NAD+-dependent pathways and receptor signaling

What are the most promising methodological approaches for targeting visfatin in translational research?

Translational approaches targeting visfatin include:

Therapeutic Modalities:

• Neutralizing antibodies: Successfully used to block visfatin-enhanced osteoclastogenesis

• Small molecule NAMPT inhibitors: Target enzymatic function

• Insulin sensitizers: Metformin modulates visfatin expression through AMPK/SIRT1 pathways

• Dietary interventions: Anthocyanins studied for effects on serum visfatin in prediabetes

Target Disease Contexts:

• Metabolic syndrome: Genetic variants strongly associated with disease risk (ORs >129)

• Bone disorders: RANKL/visfatin axis in osteoclastogenesis

• Reproductive disorders: Effects on steroidogenesis and granulosa cell function

• Inflammatory conditions: Cytokine-like properties in immune regulation

Biomarker Applications:

• Genetic screening for high-risk visfatin SNPs

• Monitoring serum visfatin in response to therapeutic interventions

• Tissue-specific expression analysis in disease states

• Combination with other metabolic and inflammatory markers