Recombinant Human Insulin-like growth factor I (IGF1) (Active) (GMP)

What is the molecular structure of recombinant human IGF-I and how does it compare to native IGF-I?

Recombinant human IGF-I is a globular protein containing 70 amino acids with a calculated molecular weight of 7.6 kDa. It contains three intramolecular disulfide bonds that are critical for maintaining its three-dimensional structure and biological activity . The amino acid sequence and structural properties of recombinant human IGF-I are identical to those of endogenous human IGF-I, making it suitable for experimental research applications. Mature human IGF-I also demonstrates 100% homology with bovine and porcine proteins, which has important implications for comparative studies across species .

When designing experiments with recombinant IGF-I, researchers should consider that proper storage and handling conditions are essential to maintain the integrity of disulfide bonds and prevent protein aggregation. Most commercially available preparations require reconstitution according to manufacturer specifications to ensure optimal biological activity.

How does IGF-I differ functionally from insulin and growth hormone?

While IGF-I shares structural similarities with insulin, it demonstrates significantly higher growth-promoting activity. A key functional difference between rhIGF-I and recombinant human growth hormone (rhGH) is that rhIGF-I exhibits hypoglycemic activity similar to insulin, whereas rhGH opposes insulin action and is diabetogenic . This distinction has important implications for experimental design and potential therapeutic applications.

• IGF-I has a longer half-life than GH, making it less susceptible to fluctuations and a more stable biomarker

• IGF-I circulates bound to specific binding proteins (IGFBPs) that modulate its bioavailability and activity

• IGF-I can stimulate glucose transport at much lower concentrations than insulin, particularly in bone-derived osteoblastic cells

What are the primary cellular responses induced by IGF-I?

IGF-I induces a diverse array of cellular responses including:

• Cell proliferation

• Cell differentiation

• Cell migration

• Cell survival and anti-apoptotic effects

These effects are tissue-dependent and context-specific. For example, IGF-I stimulates the proliferation of various cell types in muscle, bone, and cartilage tissue while enhancing glucose uptake in metabolically active cells . IGF-I is also recognized as one of the most potent activators of the AKT signaling pathway, which promotes proliferation and inhibits programmed cell death .

When designing experiments to evaluate these responses, researchers should consider using multiple complementary assays to capture the full spectrum of IGF-I bioactivities, as single readouts may not reflect the complexity of IGF-I's effects on cellular physiology.

What is the molecular mechanism of IGF-I receptor activation?

The activation of the type I insulin-like growth factor receptor (IGF1R) follows a precise molecular sequence that begins with ligand binding. Current research indicates that in the absence of ligand, the IGF1R maintains an autoinhibited state characterized by separation of the transmembrane (TM) domains . This configuration prevents spontaneous receptor activation.

The molecular activation process occurs as follows:

• IGF-I binds specifically to the α subunit of IGF1R on the cell surface

• Ligand binding induces a conformational change in the β subunit

• This conformational change releases constraints that normally keep the TMs separated

• The TMs subsequently associate, enabling the intrinsic propensity of the kinase regions to autophosphorylate

• Autophosphorylation of the receptor triggers activation of receptor tyrosine kinase activity

Molecular dynamics simulations have revealed that IGF1R TMs can form stable dimers where the two TM helices cross each other opposite a conserved proline residue (P911) . This structural insight provides important mechanistic understanding of how ligand binding translates to receptor activation across the membrane.

What are the critical downstream signaling pathways activated by IGF-I and how are they experimentally distinguished?

IGF-I receptor activation initiates several distinct but interconnected signaling cascades. The activated receptor phosphorylates multiple substrates, primarily insulin receptor substrates (IRSs) and Src homology collagen (SHC) . These phosphorylated substrates serve as docking sites for SH2 domain-containing signaling molecules, including:

• The 85 kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase)

• Growth factor receptor-bound 2 (GRB2)

• SH2-containing protein tyrosine phosphatase 2 (SHP2/Syp)

These interactions lead to the activation of two primary downstream signaling pathways:

To experimentally distinguish between these pathways, researchers commonly employ specific inhibitors (e.g., wortmannin for PI3K, U0126 for MEK), genetic knockdown using siRNA techniques, or genetic knockout via conventional methods or CRISPR-Cas9 systems . These approaches allow for the assessment of necessity or sufficiency of each pathway for specific IGF-I bioactivities.

How do IGF binding proteins (IGFBPs) modulate IGF-I activity and how should this be accounted for in experimental designs?

In circulation, IGF-I predominantly binds to specific binding proteins (IGFBPs) which serve two critical functions: prolonging the half-life of IGF-I and facilitating its delivery to target tissues . This binding substantially impacts both the bioavailability and activity of IGF-I, creating an additional layer of regulation that must be considered in experimental designs.

The presence of IGFBPs creates significant challenges for IGF-I measurement and activity assessment:

• IGFBPs can interfere with IGF-I immunoassays, potentially leading to inaccurate quantification

• The IGFBP binding affects the free fraction of IGF-I available for receptor activation

• Some IGFBPs have intrinsic biological activities independent of IGF-I

To account for these factors in experimental designs, researchers should consider:

• Using techniques that separate IGF-I from its binding proteins prior to measurement

• Implementing modern mass spectrometric methods that minimize interference from IGF-I variants

• Including controls that assess both total and free IGF-I levels

• When using recombinant IGF-I in cell culture systems, accounting for the presence of IGFBPs secreted by the cells themselves

These considerations are essential for accurate interpretation of IGF-I-related experimental data, particularly when translating between in vitro and in vivo systems.

What are the challenges in standardizing IGF-I measurement methods and how can researchers address them?

Despite its importance as a biomarker, IGF-I measurement faces significant standardization challenges that researchers must address to ensure reliable and comparable results. Key challenges include:

• Interference from IGFBPs in immunoassays

• Presence of IGF-I variants that may affect quantification

• Variability in reference intervals across different assay platforms

• Pre-analytical factors that influence IGF-I levels

To address these challenges, researchers should implement the following methodological approaches:

• Employ techniques that efficiently separate IGF-I from IGFBPs before measurement

• Consider using mass spectrometric methods that offer higher specificity and can minimize interference from IGF-I variants

• Establish assay-specific reference intervals through multicenter collaboration

• Standardize pre-analytical handling procedures including sample collection, processing, and storage conditions

The development of mass spectrometric methods has been particularly valuable for delivering more robust and accurate IGF-I measurements, as these techniques rely on precise mass determinations rather than antibody recognition . This approach also enables potential detection of pathogenic mutations through protein sequence analysis.

How should researchers design experiments to differentiate between IGF-I-specific effects and insulin-mediated effects?

Differentiating between IGF-I-specific and insulin-mediated effects presents a significant experimental challenge due to their structural similarities and overlapping signaling pathways. To address this challenge, researchers should incorporate the following design elements:

• Dose-response analyses: IGF-I stimulates glucose transport at much lower concentrations than insulin, allowing for dose-dependent discrimination

• Receptor-specific approaches:

  • Use receptor-specific blocking antibodies

  • Implement cell models with knockout or knockdown of either IGF1R or insulin receptor

  • Employ receptor-specific ligand analogs with modified binding preferences

• Downstream signaling analysis: While both hormones activate PI3K and MAPK pathways, the duration, magnitude, and specific phosphorylation patterns often differ

• Tissue-specific considerations: The relative expression levels of IGF1R versus insulin receptors vary across tissues, which can be leveraged to distinguish specific effects

When interpreting results, researchers should consider that the IGF1R and insulin receptor can form hybrid receptors with distinct signaling properties, further complicating the delineation of hormone-specific effects.

What are the optimal conditions for maintaining the stability and activity of recombinant human IGF-I in experimental settings?

Maintaining the stability and activity of recombinant human IGF-I is critical for ensuring reproducible and reliable experimental results. The following considerations should guide handling procedures:

• Storage conditions:

  • Most commercial preparations are shipped at ambient temperature but require specific storage conditions (typically -20°C or -80°C) for long-term stability

  • Avoid repeated freeze-thaw cycles, which can lead to protein degradation and activity loss

• Reconstitution protocols:

  • Follow manufacturer-specific reconstitution guidelines, typically using sterile buffers

  • Reconstituted IGF-I should be stored according to lot-specific recommendations, as outlined in the Certificate of Analysis

• Experimental media considerations:

  • Cell culture media composition can significantly affect IGF-I stability

  • Serum proteins, particularly IGFBPs secreted by cells, may bind exogenously added IGF-I

  • Consider using defined serum-free media for experiments requiring precise IGF-I dosing

• Activity verification:

  • Periodically assess the biological activity of stored IGF-I using established bioassays

  • Phosphorylation of AKT serves as a reliable indicator of IGF-I activity in cellular systems

Proper documentation of storage conditions, reconstitution protocols, and lot numbers is essential for experimental reproducibility and troubleshooting potential variations in biological responses.

What is the diagnostic utility of IGF-I measurements in growth hormone deficiency (GHD) research?

The diagnostic utility of IGF-I measurements in GHD research has been extensively investigated, with important limitations identified. Recent evidence indicates that IGF-I level has poor diagnostic accuracy as a standalone screening test for GHD . A comprehensive evaluation revealed that:

• The mean IGF-I standard deviation (SD) was not significantly different between GHD and non-GHD groups (p = 0.23)

• Receiver operating characteristic curve analysis demonstrated the best diagnostic accuracy at an IGF-I cutoff of -1.493 SD, with:

  • 0.685 sensitivity

  • 0.417 specificity

  • 0.25 positive predictive value

  • 0.823 negative predictive value

  • 0.517 area under the curve

These findings suggest that while IGF-I measurements have value in the broader diagnostic workup for GHD, they should not be used in isolation. Researchers investigating GHD should implement a multimodal diagnostic approach that includes:

• Growth hormone stimulation tests using multiple secretagogues (e.g., clonidine, arginine, L-dopa)

• Clinical evaluation of growth parameters and velocity

• IGF-I measurements as a complementary biomarker

• Consideration of additional factors that might influence IGF-I levels independent of GH status

This comprehensive approach provides more reliable diagnostic information than relying solely on IGF-I measurements.

How can researchers evaluate the efficacy and safety of recombinant human IGF-I in therapeutic applications?

Evaluating the efficacy and safety of recombinant human IGF-I in therapeutic applications requires comprehensive assessment protocols that address both short-term pharmacological effects and long-term safety considerations. Based on existing research, the following methodological framework is recommended:

• Efficacy evaluation metrics:

  • For IGF deficiency (IGFD) and short stature: growth velocity, height standard deviation scores, and body composition measurements

  • For diabetes mellitus: glycemic control parameters (HbA1c, fasting glucose, glucose tolerance)

  • Biomarker responses: IGF-I levels, IGFBP profile changes, and metabolic parameters

• Safety monitoring parameters:

  • Hypoglycemia assessment (frequency, severity, timing relative to administration)

  • Cardiovascular parameters (blood pressure, heart rate, echocardiographic measurements)

  • Cancer risk surveillance (particularly relevant given IGF-I's role in cell proliferation)

  • Specific attention to tissues with high IGF1R expression

• Comparative safety assessment:

  • Unlike rhGH which is diabetogenic, rhIGF-I has hypoglycemic activity similar to insulin, necessitating different monitoring approaches

  • Despite distinct mechanisms, rhIGF-I and rhGH share many safety considerations since IGF-I mediates most GH actions

• Study design considerations:

  • Long-term follow-up is essential given the growth-promoting and potential carcinogenic effects

  • Control groups should be carefully selected to match for underlying conditions

  • Dose-finding studies should precede efficacy trials to identify optimal therapeutic windows

This structured approach facilitates comprehensive evaluation of both therapeutic benefits and potential risks associated with recombinant human IGF-I intervention.

What are the most reliable biomarkers for monitoring IGF-I signaling pathway activation in clinical research?

Monitoring IGF-I signaling pathway activation in clinical research requires reliable biomarkers that accurately reflect receptor engagement and downstream signaling events. The following biomarkers have demonstrated value in assessing IGF-I pathway activation:

• Direct IGF1R activation markers:

  • Phosphorylated IGF1R levels in accessible tissues or circulating cells

  • IGF1R/Insulin receptor hybrid receptor phosphorylation status

  • Receptor internalization and turnover rates

• Proximal signaling components:

  • Phosphorylation status of IRS proteins (particularly IRS-1 and IRS-2)

  • Activation of PI3K, indicated by phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels

  • SHC phosphorylation status

• Downstream pathway activation:

  • Phosphorylated AKT (Ser473 and Thr308) as a primary indicator of PI3K pathway activation

  • Phosphorylated ERK1/2 reflecting MAPK pathway activation

  • mTOR pathway components (p70S6K, 4E-BP1) for protein synthesis effects

• Functional outcome markers:

  • Glucose uptake in metabolically responsive tissues

  • Protein synthesis rates using labeled amino acid incorporation

  • Gene expression profiles of IGF-I-responsive genes

When implementing these biomarkers in clinical research, consideration must be given to tissue accessibility, the temporal dynamics of signaling events, and the potential influence of other signaling pathways. Integration of multiple biomarkers typically provides more reliable assessment of pathway activation than reliance on any single marker.

What emerging technologies are advancing our understanding of IGF-I signaling dynamics?

The field of IGF-I research continues to evolve with several emerging technologies that promise to enhance our understanding of signaling dynamics and regulatory mechanisms. Key technological advances include:

• Single-cell analysis techniques that reveal cell-to-cell variability in IGF-I responses, providing insights into heterogeneous tissue responses

• Advanced protein structure determination methods such as cryo-electron microscopy, which have enabled more detailed visualization of IGF1R conformational changes during activation

• Molecular dynamics simulations that model TM associations and receptor conformational changes, revealing previously unappreciated aspects of receptor activation mechanisms

• CRISPR-Cas9 genome editing for precise manipulation of IGF-I pathway components, enabling more sophisticated functional analyses than traditional knockout approaches

• Mass spectrometry-based proteomics for comprehensive characterization of signaling networks and post-translational modifications

These technological advances are collectively enhancing our ability to dissect the complex regulatory mechanisms governing IGF-I signaling and its physiological impacts. Researchers entering this field should consider incorporating these advanced approaches to address persistent questions about receptor specificity, signaling dynamics, and context-dependent cellular responses.

What are the current challenges in developing predictive biomarkers for IGF-I responsiveness in clinical settings?

Despite advances in IGF-I research, significant challenges remain in developing predictive biomarkers for IGF-I responsiveness in clinical settings. Current limitations and research needs include:

• The poor diagnostic accuracy of IGF-I as a standalone biomarker for conditions like GHD suggests that more sophisticated biomarker panels are needed

• Variability in reference intervals across different assay platforms complicates standardization and clinical interpretation

• Interactions between IGF-I and its binding proteins create analytical challenges that affect measurement accuracy

• Individual genetic and physiological factors may significantly modulate IGF-I responsiveness independent of circulating levels

Future research should focus on:

• Developing multiparametric biomarker approaches that combine IGF-I levels with additional markers

• Implementing advanced analytical methods like mass spectrometry to enhance measurement precision

• Identifying genetic markers that predict individual responsiveness to IGF-I

• Establishing tissue-specific biomarkers that reflect local IGF-I activity rather than solely relying on systemic measurements