IGFBP7 Human, His

Basic Research Questions

• What is IGFBP7 and how does it differ from other IGFBPs?

  IGFBP7 (also known as IGFBP-rP1, MAC25, PSF, or TAF) is a secreted protein belonging to the insulin-like growth factor binding protein family. Unlike other IGFBPs, IGFBP7 exhibits higher binding affinity for insulin than for IGF-1 or IGF-2 . This distinctive binding profile makes it particularly relevant in insulin-related pathologies. The human IGFBP7 protein consists of 282 amino acids, with amino acids 27-282 representing the mature protein after signal peptide removal .

  The protein stands apart from other family members due to its significant involvement in autocrine and paracrine β-cell regulation . Additionally, it has been shown to enhance insulin action at insulin receptors in liver tissues and can interact with the IGF-1 receptor, both belonging to the Receptor tyrosine kinase (RTK) family .

• What is the significance of His-tagged IGFBP7 in experimental research?

  Histidine-tagged (His-tagged) IGFBP7 provides several methodological advantages in research applications:

  • Simplified purification: The His-tag (typically 6xHis) allows for efficient single-step purification using metal affinity chromatography

  • Enhanced detection: The tag facilitates protein tracking in complex biological systems

  • Controlled immobilization: His-tagged proteins can be directionally attached to surfaces for binding studies

  • Verification of recombinant expression: The tag serves as a marker to confirm successful protein production

  Recombinant IGFBP7 with C-terminal tags such as Avi-His-Tag can be enzymatically biotinylated, allowing for additional experimental applications like biotin-avidin pulldown assays . This dual tagging strategy enables researchers to conduct more sophisticated interaction studies and increases the versatility of the protein for various experimental designs.

• What expression patterns does IGFBP7 show in normal and pathological tissues?

  IGFBP7 shows distinctive expression patterns that vary by tissue type and disease state:

  In pancreatic islets:

  • IGFBP7 is expressed in both α- and β-cells, with higher expression observed in islets from donors with type 2 diabetes (T2D)

  • Male donors show higher baseline IGFBP7 expression compared to female donors

  • Single-cell RNA-seq analysis reveals that IGFBP7 expression is upregulated by approximately 30% in α-cells from T2D donors compared to controls, while β-cell expression remains relatively unchanged

  • IGFBP7 is highly expressed in pancreatic ductal cells from non-diabetic donors but shows approximately 70% reduction in ductal cells from T2D donors

  In cardiovascular tissue:

  • Cardiac and blood expression of IGFBP7 is robustly increased in patients with chronic heart failure

  • Similar upregulation is observed in heart failure mouse models

  These expression patterns suggest tissue-specific regulation of IGFBP7 and potential involvement in multiple pathological processes.

Intermediate Research Questions

• How can researchers effectively manipulate IGFBP7 expression in experimental models?

  Several approaches have proven effective for manipulating IGFBP7 expression in various experimental models:

  For knockdown studies:

  • siRNA transfection: Demonstrated to achieve approximately 90% knockdown in EndoC-βH1 cells (a human β-cell line)

  • shRNA delivery: AAV9-shRNA-mediated cardiac myocyte Igfbp7 knockdown has been successfully used to investigate myocardial IGFBP7's role in pathological cardiac remodeling

  For overexpression or functional studies:

  • Recombinant protein addition: Treating human islets with 100 nM IGFBP7 for 72 hours has demonstrated functional effects on insulin secretion

  • In vivo neutralization: Antibody-mediated IGFBP7 neutralization has been shown to reverse IGFBP7-induced suppression of downstream signaling pathways in cardiac models

  For genetic models:

  • Igfbp7 knockout/deficient mice have been utilized to study the protein's role in pressure overload heart failure models, revealing protective effects against cardiac dysfunction through reduced inflammation, fibrosis, and cellular senescence

  These methodological approaches provide researchers with multiple options for investigating IGFBP7 function across different experimental systems and disease models.

• What are the recommended methods for assessing IGFBP7-related functional outcomes?

  To comprehensively evaluate IGFBP7's functional effects, researchers should consider multiple complementary approaches:

  For insulin secretion studies:

  • Glucose-stimulated insulin secretion (GSIS) assays in isolated islets or β-cell lines

  • Measurement of insulin content to normalize secretion data

  • RNA-seq analysis to identify downstream gene expression changes

  For mitochondrial function assessment:

  • Oxygen consumption rate (OCR) measurements

  • ATP production assays

  • Mitochondrial membrane potential measurements

  For cardiovascular applications:

  • Evaluation of cardiac inflammatory injury markers

  • Assessment of tissue fibrosis

  • Cellular senescence markers

  • DNA repair pathway components

  • ROS detoxification signals

  For protein-protein interactions:

  • Biotin-avidin pulldown assays (particularly useful with biotin-labeled recombinant IGFBP7)

  • Co-immunoprecipitation studies

  • Binding affinity measurements with insulin, IGF-1, and their receptors

  For cellular localization:

  • Immunostaining of tissue sections with co-localization analysis (e.g., IGFBP7 with insulin or glucagon in pancreatic sections)

  • Development of image processing pipelines using guided machine learning to analyze staining intensities

  These methodological approaches provide a comprehensive toolkit for investigating IGFBP7's diverse functions in different experimental contexts.

Advanced Research Questions

• What molecular mechanisms underlie IGFBP7's inhibition of insulin secretion in pancreatic β-cells?

  IGFBP7 inhibits insulin secretion through multiple interconnected mechanisms:

  Primary molecular pathways:

  • Reduction of p21-activated kinase 1 (PAK1) protein expression

  • Decreased oxygen consumption and ATP production in β-cells

  • Impaired mitochondrial function, which is critical for glucose-stimulated insulin secretion

  Proposed mechanistic model:

  1. IGFBP7 is released locally from islet cells (demonstrated by knockdown experiments showing decreased IGFBP7 in culture medium)

  2. Extracellular IGFBP7 binds to receptors on β-cells

  3. This interaction reduces PAK1 expression

  4. Reduced PAK1 leads to impaired mitochondrial function

  5. Diminished ATP:ADP ratio in response to glucose results in decreased insulin secretion

  Acute vs. chronic effects:

  • Acute exposure to IGFBP7 also impairs glucose-stimulated insulin secretion, but likely through different mechanisms

  • Acute effects may involve RTK-dependent pathways, potentially including opening of KATP channels (similar to effects seen with hyperstimulation of insulin receptors)

  The identification of these mechanisms suggests potential therapeutic targeting strategies for improving insulin secretion in type 2 diabetes.

• How does IGFBP7 promote cellular senescence in cardiac tissue and what pathways mediate this effect?

  IGFBP7 promotes cardiac senescence through a complex signaling cascade:

  Signaling pathway:

  • IGFBP7 stimulates IGF-1R/IRS/AKT-dependent suppression of FOXO3a

  • This suppression prevents effective DNA repair mechanisms

  • It also impairs reactive oxygen species (ROS) detoxification

  • These combined effects accelerate cellular senescence and heart failure progression

  Experimental evidence:

  • In pressure overload mouse models of heart failure, Igfbp7 deficiency attenuated cardiac dysfunction

  • This protective effect occurred through reduced cardiac inflammatory injury, tissue fibrosis, and cellular senescence

  • Antibody-mediated IGFBP7 neutralization reversed IGFBP7-induced suppression of FOXO3a

  • This intervention restored DNA repair and ROS detoxification signals

  • It also attenuated pressure-overload-induced heart failure in mice

  Clinical relevance:

  • Cardiac and blood expression of IGFBP7 is robustly increased in patients with chronic heart failure

  • This suggests IGFBP7 could serve as both a biomarker and therapeutic target

  • Selectively targeting IGFBP7-regulated senescence pathways may have broad therapeutic potential for heart failure

  This mechanistic understanding positions IGFBP7 as a central regulator of age-related cardiac decline and potential therapeutic target for heart failure.

• What experimental approaches can determine if IGFBP7 is a viable therapeutic target in metabolic and cardiovascular diseases?

  Several experimental approaches can establish IGFBP7's therapeutic potential:

  For diabetes/metabolic applications:

  • siRNA knockdown of IGFBP7 in islets from T2D/IGT donors has demonstrated improved insulin secretion, supporting IGFBP7 as a potential drug target

  • Development of small molecule inhibitors that disrupt IGFBP7-receptor interactions

  • Creation of neutralizing antibodies specifically targeting IGFBP7

  • Testing IGFBP7-targeting compounds in relevant animal models of diabetes

  For cardiovascular applications:

  • Antibody-mediated IGFBP7 neutralization has already shown promising results in attenuating pressure-overload-induced heart failure in mice

  • AAV9-shRNA-mediated cardiac myocyte Igfbp7 knockdown has demonstrated that myocardial IGFBP7 directly regulates pathological cardiac remodeling

  • Long-term studies in larger animal models are needed to confirm sustained benefits

  Translational considerations:

  • Development of tissue-specific delivery methods to target IGFBP7 in pancreatic islets or cardiac tissue

  • Evaluation of potential off-target effects, given IGFBP7's expression in multiple tissues

  • Assessment of IGFBP7 as a biomarker to identify patients most likely to benefit from targeted therapies

  • Exploration of combination therapies targeting multiple aspects of disease pathophysiology

  These approaches provide a roadmap for advancing IGFBP7-targeted therapeutics from preclinical research toward potential clinical applications.