HB-EGF Rat

What is HB-EGF and what is its role in rat pancreatic β-cells?

HB-EGF is synthesized as a membrane-anchored precursor (proHB-EGF) that undergoes processing by a disintegrin and metalloproteinase (ADAM) to release the soluble active form. This growth factor plays a crucial role in pancreatic β-cell proliferation and mass regulation in rats . Research demonstrates that exposure of isolated rat islets to HB-EGF stimulates β-cell proliferation through activation of the EGFR signaling pathway .

The significance of HB-EGF extends beyond simple growth stimulation, as it forms part of a glucose/HB-EGF/EGFR axis that controls β-cell proliferation in response to metabolic demands . This pathway is essential for β-cell compensation during metabolic stress conditions, highlighting its importance in maintaining glucose homeostasis by transducing signals that increase β-cell proliferation and mass .

How does glucose regulate HB-EGF expression in rat β-cells?

Glucose regulation of HB-EGF occurs through multiple mechanisms. Experimentally, isolated rat islets exposed to 16.7 mmol/L glucose for 24 hours display a 1.5-fold increase in HB-EGF mRNA compared with islets exposed to 2.8 mmol/L glucose . This increase is primarily observed in β-cells, as demonstrated using transgenic rats expressing YFP under the control of the Ins2 promoter (RIP7-RLuc-YFP) to isolate β-cell populations by flow cytometry .

At the molecular level, glucose stimulates HB-EGF gene expression in rat β-cells through the action of carbohydrate-response element–binding protein (ChREBP) . Chromatin immunoprecipitation studies have identified ChREBP binding sites in proximity to the HB-EGF gene, providing direct evidence for ChREBP-mediated transcriptional regulation . Additionally, glucose appears to promote Src-dependent proHB-EGF processing, leading to HB-EGF shedding and stimulation of β-cell proliferation via paracrine and/or autocrine signaling through EGFR .

What experimental models are used to study HB-EGF function in rat β-cells?

Several experimental models have been developed to study HB-EGF function in rat pancreatic β-cells:

• Isolated islet culture models: Rat islets are isolated and exposed to different concentrations of glucose (typically 2.8 mmol/L vs. 16.7 mmol/L) with or without exogenous HB-EGF (100 ng/mL) . This allows for the direct assessment of HB-EGF effects on β-cell proliferation using proliferation markers such as Ki67, EdU, or pH3 combined with β-cell markers (Insulin or Nkx6.1) .

• Genetic manipulation models: Adenoviral vectors expressing short hairpin RNA targeting HB-EGF (Adv-shHBEGF) are used to knockdown HB-EGF expression in isolated rat islets . Control islets are infected with adenoviruses expressing a non-targeting shRNA (Adv-shCTL) .

• Islet transplantation models: HB-EGF-knockdown islets are transplanted under the kidney capsule of Lewis rats, followed by glucose infusion for 72 hours . This approach allows for the assessment of HB-EGF's role in glucose-induced β-cell proliferation in vivo while maintaining physiological context .

• Pharmacological inhibition models: The HB-EGF inhibitor CRM197 (10 μg/mL) or the EGFR tyrosine kinase inhibitor AG1478 (300 nmol/L) are used to block HB-EGF/EGFR signaling in isolated rat islets .

These diverse models enable comprehensive analysis of HB-EGF expression and function at both molecular and cellular levels in rat β-cells under various experimental conditions.

How does HB-EGF stimulate rat β-cell proliferation?

HB-EGF stimulates rat β-cell proliferation through a well-defined signaling cascade. When isolated rat islets are exposed to 100 ng/mL HB-EGF for 72 hours, the percentage of Ki67-positive β-cells increases to levels comparable to those detected in response to 16.7 mmol/L glucose . Similar proliferation increases are observed when using EdU as a proliferative marker .

The proliferative effect occurs via the EGFR-mTOR signaling pathway. HB-EGF induces phosphorylation of EGFR and subsequent activation of downstream signaling cascades, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT . Supporting this mechanism, exposing islets to the EGFR tyrosine kinase inhibitor AG1478 (300 nmol/L) or the mTOR complex 1 inhibitor rapamycin (10 nmol/L) completely abrogates HB-EGF–induced β-cell proliferation .

Interestingly, despite its effects on proliferation, neither acute nor prolonged (24-h) exposure to HB-EGF significantly affects insulin secretion or insulin content in rat islets ex vivo, suggesting a specific role in proliferation rather than function .

What is the difference between proHB-EGF and soluble HB-EGF in rat models?

ProHB-EGF and soluble HB-EGF represent different forms with distinct functions in rat models:

The conversion from proHB-EGF to soluble HB-EGF appears to be regulated by glucose through Src-dependent activation of metalloproteases . This is supported by research showing that Src inhibition blocked glucose-induced but not HB-EGF-induced β-cell proliferation, suggesting that glucose promotes Src-dependent proHB-EGF processing . This conversion process represents an important regulatory step in the glucose/HB-EGF/EGFR axis that controls β-cell proliferation in response to metabolic demand .

How does ChREBP regulate HB-EGF gene expression in rat β-cells under high glucose conditions?

ChREBP (carbohydrate-response element–binding protein) plays a critical role in regulating HB-EGF gene expression in rat β-cells under high glucose conditions. Experimental evidence demonstrates that ChREBP knockdown in isolated rat islets prevents glucose-induced increases in HB-EGF mRNA levels, indicating that ChREBP is necessary for glucose-stimulated HB-EGF gene expression .

Mechanistically, ChREBP functions as a glucose-responsive transcription factor that translocates to the nucleus in response to high glucose levels, where it binds to carbohydrate response elements (ChoREs) in the promoters of target genes. Chromatin immunoprecipitation studies have identified specific ChREBP binding sites in proximity to the HB-EGF gene, providing direct evidence for ChREBP-mediated transcriptional regulation .

Within the broader glucose-responsive transcriptional network, primary targets of ChREBP in the β-cell include RORγ and Myc, with HB-EGF appearing to be another direct target . Downstream of ChREBP, the HB-EGF/EGFR signaling pathway appears to work alongside these first-phase transcription factors to drive cell cycle regulators and initiate β-cell cycle progression in response to glucose . This places HB-EGF as an important mediator in the glucose-responsive transcriptional network that regulates β-cell proliferation and adaptation to metabolic demands.

What are the mechanisms of proHB-EGF processing and shedding in glucose-stimulated rat β-cells?

ProHB-EGF processing and shedding in glucose-stimulated rat β-cells involves several coordinated molecular events. The membrane-anchored proHB-EGF undergoes proteolytic cleavage by a disintegrin and metalloproteinase (ADAM) family proteins, releasing the soluble active form of HB-EGF from the cell membrane .

This process is regulated by Src family kinases in glucose-stimulated β-cells. Research demonstrates that Src inhibition blocks glucose-induced β-cell proliferation but not HB-EGF-induced proliferation, suggesting that Src acts upstream of HB-EGF shedding in the glucose response pathway . The mechanism appears similar to observations in mesangial cells, where glucose promotes HB-EGF shedding and EGFR transactivation through Src-dependent activation of metalloproteases .

Supporting this model, short-term exposure of MIN6 cells and human islets to glucose leads to phosphorylation of the Src family kinase YES . Based on these findings, researchers propose a model in which glucose activates Src family kinases, which then promote the proteolytic processing of membrane-bound proHB-EGF by ADAM metalloproteases, resulting in the release of soluble HB-EGF . This represents an important mechanism linking glucose sensing to activation of growth factor signaling.

How do Src family kinases contribute to glucose-induced HB-EGF activation in rat models?

Src family kinases serve as crucial intermediaries between glucose sensing and HB-EGF activation in rat β-cells. Experimental evidence demonstrates that inhibition of Src family kinases blocks glucose-induced β-cell proliferation but does not affect proliferation stimulated by exogenous HB-EGF . This positioning indicates that Src kinases function upstream of HB-EGF in the glucose-stimulated proliferation pathway.

The proposed mechanism involves several steps:

• Glucose stimulation activates Src family kinases in β-cells

• Activated Src kinases promote the proteolytic processing of membrane-bound proHB-EGF by ADAM metalloproteases

• This processing results in the release of soluble HB-EGF from the cell membrane

• Released HB-EGF then binds to and activates EGFR on the same cell (autocrine) or neighboring cells (paracrine)

• EGFR activation initiates downstream signaling pathways leading to β-cell proliferation

Supporting this model, research shows that short-term exposure of MIN6 cells and human islets to glucose leads to phosphorylation of the Src family kinase YES . The pathway represents an important mechanism by which glucose can rapidly activate growth factor signaling in β-cells, allowing them to respond to increased metabolic demand by expanding their mass .

What techniques can be used to specifically knock down HB-EGF in rat islets for transplantation studies?

For transplantation studies investigating HB-EGF function in rat islets, researchers have developed specific techniques for targeted knockdown:

Adenoviral-mediated RNA interference protocol:

• Islet isolation and viral transduction:

  • Isolate rat islets using standard collagenase digestion protocols

  • Culture isolated islets for 24 hours to allow recovery from isolation stress

  • Infect islets with adenoviruses expressing short hairpin RNA targeting HB-EGF (Adv-shHBEGF) or control non-targeting shRNA (Adv-shCTL)

  • Culture infected islets for an additional 24-48 hours to allow for gene knockdown

• Transplantation procedure:

  • Anesthetize recipient Lewis rats following standard protocols

  • Create a small incision to expose the kidney

  • Create a pocket under the kidney capsule

  • Transplant the infected islets (Adv-shHBEGF or Adv-shCTL) into the subcapsular space

  • Close the incision and allow rats to recover

• Glucose infusion and analysis:

  • After recovery, implant catheters for glucose or saline infusion

  • Infuse rats with glucose or saline for 72 hours

  • Harvest the kidney containing the islet graft and the endogenous pancreas

  • Analyze β-cell proliferation using immunohistochemistry for Ki67 and insulin

This approach allows researchers to specifically examine the role of HB-EGF in glucose-induced β-cell proliferation in vivo while maintaining the normal physiological context . The technique successfully demonstrated that HB-EGF knockdown in transplanted islets prevented glucose-induced β-cell proliferation, while the endogenous pancreatic islets (with normal HB-EGF expression) showed the expected increase in proliferation .

How does the glucose/HB-EGF/EGFR axis compare between rat models and human islets?

The glucose/HB-EGF/EGFR axis shows both similarities and differences between rat models and human islets:

What challenges exist in measuring HB-EGF activity in isolated rat islets versus in vivo models?

Researchers face distinct challenges when measuring HB-EGF activity in isolated rat islets compared to in vivo models:

Challenges in isolated islet studies:

• Isolation-induced stress responses:

  • The process of islet isolation induces stress that may alter gene expression patterns

  • Stress responses can affect HB-EGF expression, processing, or signaling pathways

  • Recovery periods (typically 24-48 hours) are needed before experimental interventions

• Architectural and microenvironmental changes:

  • Loss of vasculature and neural connections that normally influence islet function

  • Disruption of islet-acinar and islet-ductal cell interactions

  • Altered oxygen and nutrient gradients within isolated islets

  • These changes may affect paracrine and autocrine signaling networks relevant to HB-EGF function

• Technical limitations:

  • Diffusion barriers in intact islets can limit penetration of exogenous factors

  • Variability between islet preparations affects reproducibility

  • Limited viability for extended studies (generally <7 days in standard culture)

Challenges in in vivo models:

• Complex regulatory environment:

  • Multiple tissues produce HB-EGF, making it difficult to isolate islet-specific effects

  • Systemic factors influence both HB-EGF production and β-cell responses

  • Difficulty distinguishing direct effects from indirect effects mediated by other tissues

• Technical complexity:

  • Sophisticated approaches required to manipulate and measure HB-EGF specifically in islets

  • Need for transgenic models or transplantation approaches to achieve islet-specific manipulation

  • More complex analysis required to control for systemic variables

The transplantation model described in the research addresses some of these challenges by using adenoviral knockdown of HB-EGF in isolated islets followed by transplantation under the kidney capsule . This elegant approach allows for islet-specific manipulation of HB-EGF while maintaining the in vivo context for studying β-cell proliferation in response to glucose infusion.

How does HB-EGF signaling interact with other β-cell mitogenic pathways in rats?

HB-EGF signaling interacts with multiple β-cell mitogenic pathways in rats, forming part of an integrated network that regulates β-cell mass:

Interaction with glucose metabolism pathways:

• HB-EGF signaling is essential for glucose-induced β-cell proliferation, as inhibition of either EGFR or HB-EGF completely prevents glucose-induced β-cell proliferation

• Glucose activation of ChREBP leads to increased HB-EGF gene expression, creating a direct link between glucose metabolism and growth factor signaling

• Glucose also activates Src family kinases, which promote HB-EGF shedding, providing another connection between glucose sensing and EGFR activation

Downstream signaling convergence:

• HB-EGF activates EGFR and subsequent downstream signaling cascades, including MAPK and PI3K/AKT

• The mTOR pathway is implicated, as rapamycin (an mTOR complex 1 inhibitor) blocked HB-EGF-induced β-cell proliferation

• These pathways converge with other mitogenic signals at the level of cell cycle regulation

Interaction with other growth factor pathways:

• Other EGFR ligands (BTC, epiregulin, transforming growth factor-α, and EGF) also exert mitogenic effects on β-cells and are expressed in rodent islets

• BTC may act downstream of glucagon-like peptide 1 (GLP-1), suggesting potential crosstalk between incretin and EGFR signaling pathways

• These different EGFR ligands likely contribute to β-cell compensation in a context-dependent manner

The glucose/HB-EGF/EGFR axis appears to be particularly important for β-cell proliferation in response to increased metabolic demand, working alongside other mitogenic pathways to maintain glucose homeostasis through appropriate β-cell mass expansion .

What is the proposed model for glucose-induced HB-EGF activation in rat β-cell proliferation?

Based on the research findings, the following comprehensive model explains glucose-induced HB-EGF activation in rat β-cell proliferation:

• Glucose uptake and metabolism:

  • Glucose enters β-cells through GLUT2 transporters

  • Glucose metabolism generates signals that activate both transcriptional and non-transcriptional pathways

• Transcriptional regulation of HB-EGF:

  • Glucose metabolism activates ChREBP, a glucose-responsive transcription factor

  • Activated ChREBP translocates to the nucleus and binds to specific sites near the HB-EGF gene

  • This binding increases HB-EGF gene transcription, leading to increased proHB-EGF production

• ProHB-EGF processing and shedding:

  • Glucose activates Src family kinases in β-cells

  • Activated Src promotes activation of ADAM metalloproteases

  • These metalloproteases cleave membrane-bound proHB-EGF to release soluble HB-EGF

  • This process is critical, as Src inhibition blocks glucose-induced but not HB-EGF-induced proliferation

• EGFR activation and downstream signaling:

  • Released soluble HB-EGF binds to EGFR on β-cells

  • EGFR activation triggers phosphorylation and downstream signaling

  • Key downstream pathways include MAPK, PI3K/AKT, and mTOR signaling

  • These pathways ultimately lead to cell cycle activation

• Cell cycle progression:

  • HB-EGF/EGFR signaling, together with other ChREBP targets (like RORγ and Myc), drives expression of cell cycle regulators

  • This leads to β-cell cycle entry and progression

  • The result is increased β-cell proliferation and mass expansion to meet metabolic demands

This model, depicted in Figure 8 of the research paper, illustrates how glucose coordinates both transcriptional and non-transcriptional events to stimulate β-cell proliferation through the HB-EGF/EGFR signaling axis .