The molecular mechanisms of β-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced β-cell proliferation in rats is dependent on epidermal growth factor receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the β-cell proliferative response to glucose, a β-cell mitogen and key regulator of β-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates β-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks β-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in β-cells in response to glucose in a carbohydrate-response element–binding protein (ChREBP)–dependent manner. In addition, chromatin immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced β-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in β-cell compensation to increased metabolic demand.

In obesity, the maintenance of glucose homeostasis is dependent on the capacity of the pancreatic β-cell to meet the increased insulin requirements that arise due to insulin resistance. Failure of this mechanism leads to type 2 diabetes (1). Hence, understanding how the β-cell compensates for insulin resistance is a critical prerequisite to defining the pathogenesis of type 2 diabetes.

β-Cell compensation involves both an increase in the capacity to secrete insulin and an increase in mass. In adult rodents, β-cell expansion arises primarily from replication of existing β-cells (2,3). Over the last decade, modeling metabolic stress in rodents has led to the identification of an array of factors, including the insulin receptor (4), neurotransmitters (5), epidermal growth factor receptors (EGFRs) (6), serpin B1 (7), and nutrients (8) that control β-cell proliferation. Prominent among these factors, glucose controls β-cell replication in rodent (912) and human (13) islets. Glucose-induced β-cell proliferation requires glucokinase, ATP-sensitive potassium channel closure, and membrane depolarization (10,11). While several studies implicated insulin receptor signaling in glucose-induced β-cell replication (14,15), this observation has been challenged by evidence supporting a role for insulin receptor substrate 2 (IRS2), mammalian target of rapamycin (mTOR) (16), and the carbohydrate-response element–binding protein (ChREBP) (17,18). ChREBP is a glucose-sensing transcription factor that binds DNA with its partner Mlx at carbohydrate-response elements to stimulate glucose-responsive genes (19). Thus, the precise mechanisms underlying glucose-induced β-cell proliferation remain debated.

We established an in vivo model of nutrient excess in rats, in which a 72-h coinfusion of glucose and a lipid emulsion triggers a marked increase in β-cell proliferation and mass (20). Subsequent studies identified a signaling cascade involving EGFR–mTOR–FoxM1 that underlies the β-cell response to nutrient infusion (21). In support of these findings, EGFR loss-of-function prevents compensatory β-cell mass expansion in adult rodents under conditions of physiological (pregnancy) and pathophysiological (high-fat feeding) insulin resistance (6) as well as following partial pancreatectomy (22). However, the identity of the EGFR ligand mediating this effect remains unknown. In previous studies, we discovered that expression of the heparin-binding EGF-like growth factor (HB-EGF) is upregulated in islets from nutrient-infused rats and that exogenous HB-EGF stimulates replication of MIN6 cells and primary rat β-cells (21). HB-EGF is synthesized as a membrane-anchored precursor (proHB-EGF) that is processed by the action of a disintegrin and metalloproteinase (ADAM) to release the soluble active form (23). HB-EGF induces phosphorylation of EGFR and subsequent activation of a downstream signaling cascade, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT.

The aims of this study were 1) to determine the role of HB-EGF in the β-cell proliferative response to glucose in rat islets ex vivo and in vivo and 2) to investigate the mechanisms linking glucose to an HB-EGF/EGFR signaling pathway promoting β-cell proliferation.

Reagents and Solutions

RPMI 1640 and qualified FBS were from Invitrogen (Carlsbad, CA). Recombinant HB-EGF and betacellulin (BTC) were from R&D Systems (Minneapolis, MN). The HB-EGF inhibitor CRM197 and the Src family kinase inhibitor PP1 were from Sigma-Aldrich (St. Louis, MO). The EGFR tyrosine kinase inhibitor AG1478 and the mTOR complex 1 inhibitor rapamycin were from LC Laboratories (Woburn, MA). Adenoviruses expressing shRNAs against HB-EGF (Adv-shHBEGF) and control scrambled shRNA (Adv-shCTL) were from Vector Biolabs (Malvern, PA). SmartPool siRNA duplexes against rat ChREBP and control siRNA were obtained from Dharmacon (Lafeyette, CO). Primary antibodies and dilutions are listed in Supplementary Table 1.

Rat Islet Isolation and Adenoviral Infection

All procedures were approved by the Institutional Committee for the Protection of Animals at the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM). Islets were isolated from 2-month-old male Wistar or Lewis rats (Charles River Laboratories, Saint-Constant, Quebec, Canada) by collagenase digestion and dextran density-gradient centrifugation as described (24). For adenoviral infections, isolated islets were partially dissociated and then infected with 100 plaque-forming units of adenoviruses per cell overnight as described (25), after which the medium was replaced with complete medium and cultured for an additional 24 h prior to stimulation ex vivo or transplantation. To ensure that the HB-EGF knockdown was sustained for a period compatible with our ex vivo and in vivo experiments, we measured HB-EGF expression 5 days after infection. In Adv-shHBEGF–infected islets exposed to 16.7 mmol/L glucose, HB-EGF mRNA was reduced by 32 ± 8% (P < 0.05; n = 5) versus Adv-shCTL–infected islets.

Human Islets

Islets from human donors without diabetes were provided by the Alberta Diabetes Institute IsletCore and the Integrated Islet Distribution Program. The use of human islets was approved by the Institutional Ethics Committee of the CRCHUM (protocol number ND-05–035; Montreal, Quebec, Canada).

Islet Proliferation Ex Vivo

Rat islets were cultured in RPMI 1640 with 10% (v/v) qualified FBS (complete medium) for 72 h in the presence of glucose, 100 ng/mL HB-EGF, or 50 ng/mL BTC as indicated in the figure legends. 5-Ethynyl-2'-deoxyuridine (EdU) (10 μmol/L) was added as indicated. The media were changed every 24 h. At the end of treatment, islets were embedded in optimal cutting temperature (OCT) compound, frozen, sectioned at 8 μm, and mounted on Superfrost Plus slides (Life Technologies Inc., Burlington, Ontario, Canada). Sections were immunostained for insulin (Ins) or Nkx6.1 to mark β-cells and for the proliferative markers Ki67, phospho-histone H3 (pH3), or EdU (Click-iT EdU Imaging Kit; Life Technologies Inc.). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Images were acquired with a fluorescence microscope (Zeiss, Thornwood, NY). Proliferation was calculated as the percentage of double-positive Ki67+ (or pH3+ or EdU+) and Ins+ (or Nkx6.1+) cells over the total Ins+ (or Nkx6.1+) population. At least 1,500 β-cells from 7–17 individual islets were manually counted per condition.

Human islets were handpicked, washed with PBS, and dispersed in accutase (Innovative Cell Technologies, Inc., San Diego, CA) for 10 min at 37°C. At the end of the digestion, cells were washed, resuspended, and plated in 96-well plates (PerkinElmer, Waltham, MA) treated with poly-D-lysine hydrobromide (Sigma-Aldrich). After 24 h, dispersed human islets were cultured in RPMI 1640 with 1% (v/v) human albumin serum (Celprogen, Torrance, CA) for 72 h in the presence of glucose and 100 ng/mL HB-EGF as indicated in the figure legends. The medium was changed every 24 h. At the end of treatment, cells were fixed and immunostained for Ins and EdU. Images were acquired with an Operetta high-content imaging system (PerkinElmer) at ×20 magnification. Approximately 1,500 cells were manually counted per condition.

Static Incubations

Triplicate batches of 10 islets each were sequentially incubated twice with Krebs-Ringer bicarbonate solution containing 0.1% (w/v) BSA and 2.8 mmol/L glucose for 20 min at 37°C and then incubated for 1 h with 2.8 or 16.7 mmol/L glucose. Intracellular insulin content was measured following acid–alcohol extraction. Insulin was measured by radioimmunoassay using a rat insulin RIA kit (Millipore, Billerica, MA).

Islet Transplantation and Glucose Infusions in Rats

Male Lewis rats weighing 250–350 g (∼2-month-old) (Charles River Laboratories) underwent catheterization of the jugular vein for infusion and the carotid artery for sampling as described (26). For islet transplantation, 500 islets isolated from 2-month-old male Lewis rats were infected with Adv-shHBEGF or Adv-shCTL, as described above, and injected via a cannula under the left kidney capsule during the catheterization surgery. Animals were allowed to recover for 72 h followed by intravenous infusions of either saline (0.9% w/v NaCl) (Baxter, Mississauga, Ontario, Canada) or 70% (w/v) glucose (McKesson, Montreal, Quebec, Canada) for an additional 72 h. The glucose infusion rate was adjusted to maintain plasma glucose at 13.9–19.4 mmol/L throughout the 72-h infusion.

Immunostaining of Tissue Sections

Transplanted kidneys and pancreata were fixed for 4 h in 4% paraformaldehyde and cryoprotected overnight in 30% sucrose. Tissues were then embedded in OCT, frozen, sectioned at 8 μm, and mounted on Superfrost Plus slides (Life Technologies Inc.). Antigen retrieval was performed using sodium citrate buffer, and β-cell proliferation was assessed as described above.

Flow Cytometry of β-Cells

Islets were isolated from male RIP7-RLuc-YFP transgenic rats (27), washed in PBS, and dispersed in accutase for 10 min at 37°C. At the end of the digestion, cells were washed, resuspended in PBS, and passed through a 40-μm filter prior to sorting. Flow cytometric sorting of yellow fluorescent protein (YFP)–positive and –negative cells was carried out using an FACSAria II flow cytometer with FACSDiva software (BD Biosciences, San Jose, CA). YFP-expressing cells were detected using the 488-nm laser and 530/30-nm bandpass filter.

Quantitative RT-PCR

Total RNA was extracted from 150 to 200 whole islets or 100,000 sorted islet cells using the RNeasy Micro kit (Qiagen, Valencia, CA). RNA was quantified by spectrophotometry using a NanoDrop 2000 (Life Technologies Inc.), and 1 μg of RNA was reverse transcribed. Real-time PCR was performed by using the QuantiTect SYBR Green PCR kit (Qiagen). Results were normalized to cyclophilin A RNA levels.

Chromatin Immunoprecipitation and Chromatin Confirmation Capture

INS-1 832/13 cell culture, siRNA treatment, RNA isolation, and RT-PCR were performed as described (17). The pool of siRNA duplexes directed against ChREBP was previously shown to significantly decrease ChREBP mRNA (65%) and protein (70%) levels (17). Chromatin immunoprecipitation (ChIP) was performed as previously described (18). Briefly, INS-1 cells were cultured for 16 h in 2 mmol/L glucose followed by 6 h at 2 or 20 mmol/L glucose. An anti-ChREBP or normal rabbit IgG was used for immunoprecipitation, and a genomic region 30 kb downstream from the transcription start site of the HB-EGF gene known to bind ChREBP (28) was amplified by RT-PCR. Chromatin confirmation capture (3C) was performed essentially as described in Hagège et al. (29). INS-1 cells were treated as for ChIP. The sequences of primers used for RT-PCR, ChIP, and 3C are shown in Supplementary Table 2.

Immunoblotting and ELISA

For immunoblotting, proteins were extracted from rat islets and subjected to 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with primary antibodies against phospho-EGFR, phospho–S6 ribosomal protein (S6RP), and α-tubulin in 5% (w/v) milk. Signals were revealed using horseradish peroxidase–conjugated anti-rabbit IgG secondary antibodies (Bio-Rad, Hercules, CA) in 5% (w/v) milk and visualized using Western Lighting Plus-ECL (PerkinElmer). Band intensity was quantified using ImageJ software (National Institutes of Health).

HB-EGF was measured by ELISA (MyBioSource, San Diego, CA) in protein extracts from 200 to 300 rat islets treated with glucose for 1 h.

Statistical Analyses

Data are expressed as means ± SEM. Significance was tested using one-way ANOVA with Tukey or Dunnett post hoc test or two-way ANOVA with post hoc adjustment for multiple comparisons, as appropriate, using GraphPad InStat (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.

Data and Resource Availability

All data generated or analyzed during this study are included in the published article (and the Supplementary Data). No applicable resources were generated or analyzed during the current study.

HB-EGF Induces β-Cell Proliferation via EGFR–mTOR Signaling

We previously showed that HB-EGF stimulates β-cell proliferation in dispersed rat islets (21). To confirm and extend these findings, we assessed the β-cell proliferative response to HB-EGF in intact rat islets after a 72-h exposure using either Ki67 or EdU labeling to mark proliferating cells and Ins or Nkx6.1 to mark β-cells (Fig. 1). In the presence of 2.8 mmol/L glucose, 100 ng/mL HB-EGF or 50 ng/mL BTC increased the percentage of Ki67-positive β-cells to levels comparable to those detected in response to 16.7 mmol/L glucose (Fig. 1A–D). Similar results were obtained when using EdU as a proliferative marker (Fig. 1E and F). Exposing islets to the EGFR tyrosine kinase inhibitor AG1478 (300 nmol/L) or the mTOR complex 1 inhibitor rapamycin (10 nmol/L) abrogated HB-EGF–induced β-cell proliferation (Fig. 1G and H). In isolated dispersed human islets, exposure to HB-EGF for 72 h also induced β-cell proliferation (Fig. 2).

Figure 1

HB-EGF stimulates β-cell proliferation via the EGFR. AF: Isolated rat islets were exposed to 2.8 mmol/L glucose, 16.7 mmol/L glucose, or HB-EGF (100 ng/mL) or BTC (50 ng/mL) in the presence of 2.8 mmol/L glucose for 72 h. G and H: Isolated rat islets were exposed to 2.8 mmol/L glucose and left untreated or treated with HB-EGF (100 ng/mL) with or without AG1478 (300 nmol/L) or rapamycin (Rap; 10 nmol/L) for 72 h. Proliferation was assessed by Ki67 (AD, G, and H) or EdU (E and F) staining and Nkx6.1 (A and B) or Ins (CH). Representative images of Nkx6.1 (red), Ki67 (green), and nuclei (blue) (A) or Ins (green), Ki67 or EdU (red), and nuclei (blue) (C, E, and G) staining. Arrows show nuclei positive for Ki67 and EdU. B, D, and H: The percentage of Ki67+Ins+ (or Nkx6.1+) cells of total Ins+ (or Nkx6.1+) cells. F: Percentage of EdU+Ins+ cells of total Ins+ cells. Data represent individual values and are expressed as means ± SEM (n = 4–6). Scale bars, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the 2.8 mmol/L glucose control condition. ns, not significant; Veh, vehicle.

Figure 1

HB-EGF stimulates β-cell proliferation via the EGFR. AF: Isolated rat islets were exposed to 2.8 mmol/L glucose, 16.7 mmol/L glucose, or HB-EGF (100 ng/mL) or BTC (50 ng/mL) in the presence of 2.8 mmol/L glucose for 72 h. G and H: Isolated rat islets were exposed to 2.8 mmol/L glucose and left untreated or treated with HB-EGF (100 ng/mL) with or without AG1478 (300 nmol/L) or rapamycin (Rap; 10 nmol/L) for 72 h. Proliferation was assessed by Ki67 (AD, G, and H) or EdU (E and F) staining and Nkx6.1 (A and B) or Ins (CH). Representative images of Nkx6.1 (red), Ki67 (green), and nuclei (blue) (A) or Ins (green), Ki67 or EdU (red), and nuclei (blue) (C, E, and G) staining. Arrows show nuclei positive for Ki67 and EdU. B, D, and H: The percentage of Ki67+Ins+ (or Nkx6.1+) cells of total Ins+ (or Nkx6.1+) cells. F: Percentage of EdU+Ins+ cells of total Ins+ cells. Data represent individual values and are expressed as means ± SEM (n = 4–6). Scale bars, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the 2.8 mmol/L glucose control condition. ns, not significant; Veh, vehicle.

Figure 2

HB-EGF stimulates β-cell proliferation in human islets. Human islets were dispersed and then subjected to 2.8 or 16.7 mmol/L glucose or HB-EGF (100 ng/mL) in the presence of 2.8 mmol/L glucose for 72 h. Proliferation was assessed by EdU and Ins to mark β-cells. A: Representative images of Ins (green), EdU (red), and nuclei (blue) staining. Arrows show nuclei positive for EdU. B: The percentage of EdU+Ins+ cells of total Ins+ cells. Data represent individual values and are expressed as means ± SEM (n = 9). Scale bar, 50 μm. **P < 0.01 as compared with the 2.8 mmol/L glucose control condition.

Figure 2

HB-EGF stimulates β-cell proliferation in human islets. Human islets were dispersed and then subjected to 2.8 or 16.7 mmol/L glucose or HB-EGF (100 ng/mL) in the presence of 2.8 mmol/L glucose for 72 h. Proliferation was assessed by EdU and Ins to mark β-cells. A: Representative images of Ins (green), EdU (red), and nuclei (blue) staining. Arrows show nuclei positive for EdU. B: The percentage of EdU+Ins+ cells of total Ins+ cells. Data represent individual values and are expressed as means ± SEM (n = 9). Scale bar, 50 μm. **P < 0.01 as compared with the 2.8 mmol/L glucose control condition.

We then asked whether HB-EGF affects insulin secretion in rat islets. Isolated islets were exposed to HB-EGF either simultaneously with glucose during a 1-h static incubation to measure insulin secretion or during the 24-h period preceding the static incubation. Neither acute nor prolonged exposure to HB-EGF significantly affected insulin secretion or insulin content (Supplementary Fig. 1). These results indicate that exogenous HB-EGF promotes rat β-cell proliferation via EGFR and mTOR without significantly affecting insulin secretion.

Glucose-Induced β-Cell Proliferation in Isolated Rat Islets Requires HB-EGF/EGFR Signaling

Given that glucose is a known β-cell mitogen (912), we next examined the contribution of HB-EGF/EGFR signaling to glucose-induced β-cell proliferation. Treatment of rat islets for 72 h with 16.7 mmol/L glucose led to an approximately threefold increase in Ki67 staining compared with 2.8 mmol/L glucose (Fig. 3A and B). Addition of AG1478 completely prevented the glucose-induced increase in β-cell proliferation (Fig. 3A and B). Likewise, the HB-EGF inhibitor CRM197 (10 μg/mL) blocked the stimulatory effect of glucose on β-cell proliferation (Fig. 3C and D). Similar findings were obtained by labeling rat islets with Nkx6.1 and the M-phase marker pH3 after exposure to HB-EGF, 16.7 mmol/L glucose, or 16.7 mmol/L glucose+ CRM197 (Fig. 3E and F). To further substantiate the implication of HB-EGF in glucose-induced β-cell proliferation, we infected isolated rat islets with Adv-shHBEGF or Adv-shCTL (Fig. 3G and H). Following a 72-h exposure to 16.7 mmol/L glucose, Adv-shHBEGF–infected islets did not display any increase in β-cell proliferation (Fig. 3G and H). Collectively, these results demonstrate that HB-EGF/EGFR signaling is required for glucose-induced β-cell proliferation in isolated rat islets.

Figure 3

Glucose stimulates β-cell proliferation via HB-EGF/EGFR signaling. A and B: Isolated rat islets were exposed to 2.8 or 16.7 mmol/L glucose with or without AG1478 (300 nmol/L) for 72 h. C and D: Isolated rat islets were exposed to 2.8 mmol/L glucose, 16.7 mmol/L glucose, or 2.8 mmol/L glucose plus 100 ng/mL HB-EGF in the absence or presence of 10 μg/mL CRM197 for 72 h. E and F: Isolated rat islets were exposed to 2.8 mmol/L glucose with or without 100 ng/mL HB-EGF or 16.7 mmol/L glucose with or without 10 μg/mL CRM197 for 72 h. G and H: Isolated rat islets were infected with Adv-shHBEGF or Adv-shCTL and exposed to 2.8 or 16.7 mmol/L glucose for 72 h. Proliferation was assessed by Ki67 (AD, G, and H) or pH3 (E and F) staining and Ins (AD, G, H) or Nkx6.1 (E and F) staining to mark β-cells. A, C, E, and G: Representative images of Ins (green) or Nkx6.1 (red), Ki67 (red) or pH3 (green), and nuclei (blue) staining. Arrows show nuclei positive for Ki67 or pH3. B, D, and H: Percentage of Ki67+Ins+ cells of total Ins+ cells. F: Percentage of pH3+Nkx6.1+ cells of total Nkx6.1+ cells. Data represent individual values and means ± SEM (n = 4–6). Scale bars, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as compared with the 2.8 mmol/L glucose condition; &&P < 0.01 as compared with 16.7 mmol/L glucose. ns, not significant; Veh, vehicle.

Figure 3

Glucose stimulates β-cell proliferation via HB-EGF/EGFR signaling. A and B: Isolated rat islets were exposed to 2.8 or 16.7 mmol/L glucose with or without AG1478 (300 nmol/L) for 72 h. C and D: Isolated rat islets were exposed to 2.8 mmol/L glucose, 16.7 mmol/L glucose, or 2.8 mmol/L glucose plus 100 ng/mL HB-EGF in the absence or presence of 10 μg/mL CRM197 for 72 h. E and F: Isolated rat islets were exposed to 2.8 mmol/L glucose with or without 100 ng/mL HB-EGF or 16.7 mmol/L glucose with or without 10 μg/mL CRM197 for 72 h. G and H: Isolated rat islets were infected with Adv-shHBEGF or Adv-shCTL and exposed to 2.8 or 16.7 mmol/L glucose for 72 h. Proliferation was assessed by Ki67 (AD, G, and H) or pH3 (E and F) staining and Ins (AD, G, H) or Nkx6.1 (E and F) staining to mark β-cells. A, C, E, and G: Representative images of Ins (green) or Nkx6.1 (red), Ki67 (red) or pH3 (green), and nuclei (blue) staining. Arrows show nuclei positive for Ki67 or pH3. B, D, and H: Percentage of Ki67+Ins+ cells of total Ins+ cells. F: Percentage of pH3+Nkx6.1+ cells of total Nkx6.1+ cells. Data represent individual values and means ± SEM (n = 4–6). Scale bars, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as compared with the 2.8 mmol/L glucose condition; &&P < 0.01 as compared with 16.7 mmol/L glucose. ns, not significant; Veh, vehicle.

Glucose-Induced β-Cell Proliferation in Transplanted Rat Islets Requires HB-EGF

To test whether islet-derived HB-EGF is necessary for glucose-induced β-cell proliferation in vivo, islets infected with either Adv-shHBEGF or Adv-shCTL were transplanted under the kidney capsule of Lewis rats. The rats were then infused with saline or glucose for 72 h (Fig. 4A). Average blood glucose levels and glucose infusion rates were not different between both groups (Supplementary Fig. 2). As expected, the glucose infusion increased the percentage of Ki67-positive β-cells in the endogenous pancreas to the same extent in Adv-shCTL and Adv-shHBEGF transplant recipients (Fig. 4B and C). Adv-shCTL–infected islet grafts also showed increased β-cell proliferation in response to glucose infusion (Fig. 4D and E). In contrast, Adv-shHBEGF–infected islets were unresponsive to glucose (Fig. 4D and E). These data demonstrate that, as observed in isolated islets (Fig. 3), HB-EGF/EGFR signaling is required for glucose-induced β-cell proliferation in vivo.

Figure 4

HB-EGF is required for glucose (Glu)–induced β-cell proliferation in vivo. A: Isolated rat islets were infected with Adv-shHBEGF or Adv-shCTL and transplanted under the kidney capsule of 2-month-old Lewis rats infused with saline (Sal) or Glu for 72 h. BE: Proliferation was assessed by Ki67 and Ins staining. B and D: Representative images of Ins (green), Ki67 (red), and nuclei (blue) staining in the pancreas (B) or transplanted islets (D). Arrows show nuclei positive for Ki67. C and E: The percentage of Ki67+Ins+ cells of total Ins+ cells in the pancreas (C) and transplanted islets (E). Data represent individual values and means ± SEM (n = 4–6). Scale bars, 50 μm. ***P < 0.001, ****P < 0.0001 as compared with the Sal condition. ns, not significant.

Figure 4

HB-EGF is required for glucose (Glu)–induced β-cell proliferation in vivo. A: Isolated rat islets were infected with Adv-shHBEGF or Adv-shCTL and transplanted under the kidney capsule of 2-month-old Lewis rats infused with saline (Sal) or Glu for 72 h. BE: Proliferation was assessed by Ki67 and Ins staining. B and D: Representative images of Ins (green), Ki67 (red), and nuclei (blue) staining in the pancreas (B) or transplanted islets (D). Arrows show nuclei positive for Ki67. C and E: The percentage of Ki67+Ins+ cells of total Ins+ cells in the pancreas (C) and transplanted islets (E). Data represent individual values and means ± SEM (n = 4–6). Scale bars, 50 μm. ***P < 0.001, ****P < 0.0001 as compared with the Sal condition. ns, not significant.

HB-EGF Gene Expression Is Upregulated in β-Cells in Response to Glucose

As we previously showed that infusion of glucose and lipids in rats increases HB-EGF mRNA levels in islets (21), we asked whether glucose alone was sufficient to stimulate HB-EGF expression in isolated islets. Indeed, isolated rat islets exposed to 16.7 mmol/L glucose for 24 h displayed a 1.5-fold increase of HB-EGF mRNA compared with 2.8 mmol/L glucose (Fig. 5A). To determine whether the increase in islet HB-EGF gene expression was primarily in β-cells, we used a transgenic rat expressing YFP under the control of the Ins2 promoter (RIP7-RLuc-YFP) (27) to enrich for β-cells by flow cytometry after glucose treatment. Glucose augmented HB-EGF mRNA levels in the YFP-positive (β-cell enriched) (Fig. 5B) cells, but not the YFP-negative (Fig. 5C) fraction, suggesting that glucose stimulates HB-EGF gene expression in rat β-cells.

Figure 5

Glucose increases HB-EGF gene expression in the β-cell. HB-EGF mRNA was measured in isolated intact rat islets (A) or in FACS-sorted YFP-positive (B) and YFP-negative (C) cells from isolated RIP7-RLuc-YFP islets following exposure to 2.8 or 16.7 mmol/L glucose for 24 h. mRNA was determined by quantitative RT-PCR and normalized to cyclophilin A. Data are presented as the fold increase over the 2.8 mmol/L glucose condition and represent individual values and means ± SEM (n = 5 to 6). *P < 0.05 as compared with the 2.8 mmol/L glucose condition. ns, not significant.

Figure 5

Glucose increases HB-EGF gene expression in the β-cell. HB-EGF mRNA was measured in isolated intact rat islets (A) or in FACS-sorted YFP-positive (B) and YFP-negative (C) cells from isolated RIP7-RLuc-YFP islets following exposure to 2.8 or 16.7 mmol/L glucose for 24 h. mRNA was determined by quantitative RT-PCR and normalized to cyclophilin A. Data are presented as the fold increase over the 2.8 mmol/L glucose condition and represent individual values and means ± SEM (n = 5 to 6). *P < 0.05 as compared with the 2.8 mmol/L glucose condition. ns, not significant.

Glucose Stimulates HB-EGF Gene Expression via ChREBP

ChREBP is a key mediator of glucose-induced transcriptional changes (28). Therefore, we asked whether HB-EGF is a direct target of ChREBP. Consistent with the results shown in Fig. 5, glucose increased HB-EGF expression in untransfected INS-1 cells and in cells transfected with a control siRNA (Fig. 6A). In contrast, siRNA-mediated knockdown of ChREBP abolished the glucose response (Fig. 6A). ChREBP ChIP-sequencing and DNase-sequencing analyses of INS-1 cells exposed to glucose identified putative enhancer elements containing canonical ChREBP binding sites located ∼30 kb downstream of the HB-EGF transcription start site (28). ChIP analysis for one of these elements showed that a 6-h exposure to 20 mmol/L glucose significantly increased ChREBP binding, whereas binding to a control region was unchanged (Fig. 6B). Furthermore, 3C analysis revealed increased interactions between these enhancers and the HB-EGF promoter in the presence of 20 mmol/L glucose (Fig. 6C). These results show that glucose-induced HB-EGF gene expression is mediated by direct binding of ChREBP to enhancers located 3′ to the HB-EGF gene.

Figure 6

ChREBP mediates glucose-induced HB-EGF gene expression in INS-1 cells. A: HB-EGF RNA was measured in INS-1 cells exposed to 3 or 15 mmol/L glucose for 18 h in the presence of a control siRNA (SiCon) or an siRNA directed against ChREBP (SiChR) (n = 3). mRNA was determined by quantitative RT-PCR and normalized to β-actin. B: ChREBP binding to a genomic region 30 kb downstream from the transcription start site of the HB-EGF gene known to bind ChREBP (black bar in C, top panel) was assessed in INS-1 cells exposed to 2 or 20 mmol/L glucose for 6 h followed by ChIP using an antibody against ChREBP or control IgG (n = 3). Data indicate the percent binding after subtraction of the IgG control. B (inset): Pklr coding region serves as a negative control. C (top): Genome browser view of 38,000 bp of the genomic locus spanning the transcription start site (TSS) of the HB-EGF gene showing the ChREBP binding (ChREBP ChIP) and DNAse hypersensitivity sites (DNAse HS) downstream of the gene (28). Black bar is the region amplified in B. C (bottom): 3C data from INS-1 cells treated as in B, aligned to the genome browser and expressed as interaction frequency normalized to maximum interaction (n = 3). Black line, anchor primer. Shaded gray added for clarity represents interaction frequency after 20 mmol/L glucose treatment. Data are expressed as means ± SEM. *P < 0.05, ***P < 0.001 as compared with the control condition. kbp, kilobase pair; ns, not significant; NT, nontransfected.

Figure 6

ChREBP mediates glucose-induced HB-EGF gene expression in INS-1 cells. A: HB-EGF RNA was measured in INS-1 cells exposed to 3 or 15 mmol/L glucose for 18 h in the presence of a control siRNA (SiCon) or an siRNA directed against ChREBP (SiChR) (n = 3). mRNA was determined by quantitative RT-PCR and normalized to β-actin. B: ChREBP binding to a genomic region 30 kb downstream from the transcription start site of the HB-EGF gene known to bind ChREBP (black bar in C, top panel) was assessed in INS-1 cells exposed to 2 or 20 mmol/L glucose for 6 h followed by ChIP using an antibody against ChREBP or control IgG (n = 3). Data indicate the percent binding after subtraction of the IgG control. B (inset): Pklr coding region serves as a negative control. C (top): Genome browser view of 38,000 bp of the genomic locus spanning the transcription start site (TSS) of the HB-EGF gene showing the ChREBP binding (ChREBP ChIP) and DNAse hypersensitivity sites (DNAse HS) downstream of the gene (28). Black bar is the region amplified in B. C (bottom): 3C data from INS-1 cells treated as in B, aligned to the genome browser and expressed as interaction frequency normalized to maximum interaction (n = 3). Black line, anchor primer. Shaded gray added for clarity represents interaction frequency after 20 mmol/L glucose treatment. Data are expressed as means ± SEM. *P < 0.05, ***P < 0.001 as compared with the control condition. kbp, kilobase pair; ns, not significant; NT, nontransfected.

Glucose-Induced β-Cell Proliferation Is Dependent on Src Upstream of EGFR Activation, but Glucose-Induced mTOR Activation Does Not Require HB-EGF

Processing of proHB-EGF by ADAM proteins releases the active form that binds and activates EGFR (23). Previous studies in mesangial cells suggest that glucose-induced proteolytic processing of HB-EGF requires Src activation (30). Therefore, we investigated the role of Src family kinases in glucose-induced β-cell proliferation. Addition of the Src inhibitor PP1 abrogated the β-cell proliferative response to 16.7 mmol/L glucose but not to HB-EGF (Fig. 7A and B), consistent with the possibility that glucose promotes proHB-EGF cleavage via Src followed by HB-EGF activation of EGFR. To assess glucose-stimulated HB-EGF shedding, we attempted to measure HB-EGF levels in islet-conditioned media following a 1-h exposure to 16.7 mmol/L glucose. Unfortunately, HB-EGF levels in the samples were below the detection limit of the assay. However, we observed a trend toward an increase in total HB-EGF levels in islet extracts (Supplementary Fig. 3), which, although not statistically significant, is consistent with the glucose-induced HB-EGF expression shown in Figs. 5A and 6A.

Figure 7

Src is required for glucose- but not HB-EGF–induced β-cell proliferation, and glucose-induced mTOR activation does not require HB-EGF. A and B: Isolated rat islets were exposed to 2.8 or 16.7 mmol/L glucose or HB-EGF (100 ng/mL) in the presence of 2.8 mmol/L glucose for 72 h with or without the Src inhibitor PP1 (1 μmol/L). Proliferation was assessed by Ki67 staining and Ins. A: Representative images of Ins (green), Ki67 (red), and nuclei (blue). Arrows show nuclei positive for Ki67. B: The percentage of Ki67+Ins+ cells of total Ins+ cells. Scale bar, 50 μm. C and D: Isolated rat islets were exposed to 2.8 or 16.7 mmol/L glucose or HB-EGF (100 ng/mL) in the presence of 2.8 mmol/L glucose with or without CRM197 (10 μg/mL) for 24 and 48 h. Representative Western blot (C) of phospho-S6RP (pS6RP) and α-tubulin and densitometric quantification (D) of pS6RP normalized to α-tubulin. Data represent individual values and means ± SEM (n = 4–6). *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the 2.8 mmol/L glucose condition or as indicated in the graph (B). ns, not significant; Veh, vehicle.

Figure 7

Src is required for glucose- but not HB-EGF–induced β-cell proliferation, and glucose-induced mTOR activation does not require HB-EGF. A and B: Isolated rat islets were exposed to 2.8 or 16.7 mmol/L glucose or HB-EGF (100 ng/mL) in the presence of 2.8 mmol/L glucose for 72 h with or without the Src inhibitor PP1 (1 μmol/L). Proliferation was assessed by Ki67 staining and Ins. A: Representative images of Ins (green), Ki67 (red), and nuclei (blue). Arrows show nuclei positive for Ki67. B: The percentage of Ki67+Ins+ cells of total Ins+ cells. Scale bar, 50 μm. C and D: Isolated rat islets were exposed to 2.8 or 16.7 mmol/L glucose or HB-EGF (100 ng/mL) in the presence of 2.8 mmol/L glucose with or without CRM197 (10 μg/mL) for 24 and 48 h. Representative Western blot (C) of phospho-S6RP (pS6RP) and α-tubulin and densitometric quantification (D) of pS6RP normalized to α-tubulin. Data represent individual values and means ± SEM (n = 4–6). *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the 2.8 mmol/L glucose condition or as indicated in the graph (B). ns, not significant; Veh, vehicle.

Glucose-induced β-cell proliferation is dependent on mTOR activation (16). As the mitogenic effect of HB-EGF was also dependent on mTOR in rat islets (Fig. 1G and H), we asked whether mTOR activation by glucose is dependent on HB-EGF. Exposing islets for 24 and 48 h to 16.7 mmol/L glucose led to a significant increase in phosphorylation of the mTOR substrate S6RP (Fig. 7C and D). However, HB-EGF did not increase S6RP phosphorylation, and blocking HB-EGF with CRM197 did not affect glucose-induced mTOR activation (Fig. 7C and D). Hence, glucose activation of mTOR is independent of HB-EGF.

The results of this study demonstrate a critical role for HB-EGF in glucose-induced β-cell proliferation in rat β-cells. Exposing isolated islets to exogenous HB-EGF induced β-cell proliferation, whereas blocking HB-EGF signaling by inhibiting either EGFR or HB-EGF completely prevented the proliferative response. In vivo, silencing HB-EGF prevented the increase in β-cell proliferation in islets transplanted under the kidney capsule of glucose-infused rats. Taken together, our data identify a glucose/HB-EGF/EGFR axis that controls β-cell proliferation. Mechanistically, we showed that HB-EGF gene expression is induced by glucose in the β-cell through the action of ChREBP. In addition, we found that glucose-induced, but not HB-EGF–induced, β-cell proliferation is blocked by Src inhibition. As Src family kinases are involved in EGFR transactivation via ADAM metalloproteases, we propose a mechanism by which glucose activates ChREBP and Src to promote HB-EGF gene expression and HB-EGF membrane shedding, respectively, and subsequently EGFR downstream signaling and cell cycle activation (Fig. 8).

Figure 8

Proposed mechanism of glucose/HB-EGF/EGFR axis controlling β-cell proliferation. An increase in the soluble, active form of HB-EGF is mediated by glucose-induced ChREBP, which increases HB-EGF gene expression, and by glucose-induced Src, which is coupled to metalloprotease (ADAM)–dependent proHB-EGF processing. Subsequent binding of HB-EGF to the β-cell EGFR activates signaling pathways including mTOR but also possibly MAPK, PI3K/AKT, and IRS2 that together promote β-cell proliferation. Inhibitors used in this study to block glucose- and HB-EGF–induced β-cell proliferation are indicated.

Figure 8

Proposed mechanism of glucose/HB-EGF/EGFR axis controlling β-cell proliferation. An increase in the soluble, active form of HB-EGF is mediated by glucose-induced ChREBP, which increases HB-EGF gene expression, and by glucose-induced Src, which is coupled to metalloprotease (ADAM)–dependent proHB-EGF processing. Subsequent binding of HB-EGF to the β-cell EGFR activates signaling pathways including mTOR but also possibly MAPK, PI3K/AKT, and IRS2 that together promote β-cell proliferation. Inhibitors used in this study to block glucose- and HB-EGF–induced β-cell proliferation are indicated.

Our previous (21) and current results are in agreement with studies showing that overexpression of HB-EGF by retrograde injection of adenoviruses into the pancreatic duct leads to proliferation of pre-existing β-cells in adult mice (31). In contrast, no increase in β-cell proliferation was observed following HB-EGF expression in developing mouse β-cells (32). However, the presence of pancreatic fibrosis, stromal expansion, and islet dysfunction in this model may have precluded such an effect. Interestingly, overexpression of HB-EGF (31) or BTC (33) in pancreatic ducts promotes β-cell neogenesis, and EGF gain-of-function studies in human duct cells (34,35) support a similar conclusion. Hence, we propose that the major effect of HB-EGF is to promote proliferation of existing β-cells, but that β-cell neogenesis could also contribute to its overall beneficial effects on β-cell mass. In contrast to its effects on β-cell proliferation, acute and extended (24-h) exposure to HB-EGF did not alter insulin secretion or insulin content in rat islets ex vivo. However, positive, antidiabetic effects of HB-EGF on the β-cell were demonstrated in multiple low-dose streptozotocin diabetic mice by which combined treatment of gastrin and HB-EGF led to improved islet function due in part to a reduction in insulitis (36). Further studies will be required to fully elucidate the pleotropic effects of HB-EGF on pancreatic islets.

We found that AG1478, a specific inhibitor of EGFR with minimal activity toward other ErbB isoforms, completely abrogates HB-EGF–induced β-cell proliferation. As HB-EGF signals via EGFR (ErbB1) and ErbB4 but not ErbB2 or ErbB3 (37), and EGFR is expressed in β-cells, whereas ErbB4 is only weakly expressed in rodent islets (38), we propose that HB-EGF acts predominantly via EGFR to promote β-cell proliferation.

EGFR inhibition, loss-of-function, and dominant-negative studies in adult rodents in the context of pathophysiological and physiological metabolic stress (6,21,39) and partial pancreatectomy (22) suggest that β-cell EGFR underlies the maintenance of glucose homeostasis by transducing signals that increase β-cell proliferation and mass. Notwithstanding a role for BTC downstream of glucagon-like peptide 1 (40), however, attempts to investigate the role of EGFR ligands in the regulation of β-cell mass and function have been limited to gain-of-function approaches (31,33,41,42), whereas the identification of endogenous ligands contributing to β-cell compensation is unknown. We found that glucose, a key effector of regulation of β-cell mass in the face of increased insulin demand (11), requires HB-EGF signaling. When rat islets were exposed to glucose ex vivo or in vivo, the β-cell mitogenic response was dependent on both EGFR and HB-EGF. Although HB-EGF was essential for the glucose response, whether HB-EGF is the sole endogenous EGFR ligand acting during β-cell compensation to metabolic stress remains an open question. BTC (41), epiregulin (43), transforming growth factor-α, and EGF (41,44) exert mitogenic effects on the β-cell and are expressed in developing (45) and adult (38) rodent islets and during β-cell neogenesis (46). Hence, different EGFR ligands likely contribute to β-cell compensation in a context-dependent manner.

In previous studies, we showed that HB-EGF gene expression is upregulated in islets following nutrient infusion in rats (21), and a similar trend was found in obese, diabetes-resistant (B6) mice (47). Our present results suggest that the increase in HB-EGF gene expression is due, at least in part, to the direct action of glucose. They are consistent with the time- and dose-dependent increase in HB-EGF gene expression observed in response to glucose in INS-1 cells (28) and rat islets (48). In addition, we found that ChREBP is necessary for HB-EGF gene expression and that ChREBP binds a 3′ HB-EGF gene enhancer element. Primary targets of ChREBP in the β-cell include RORγ and Myc (28), whereas the cell cycle regulatory cyclins and cyclin-dependent kinases, which lack ChREBP binding sites, respond to glucose in a delayed manner due to their dependency on first-phase factors (17,28). Hence, downstream of ChREBP, HB-EGF/EGFR signaling could play a role alongside first-phase transcription factors to drive cell cycle regulators and initiate β-cell cycle progression in response to glucose (Fig. 8).

Although membrane-anchored proHB-EGF may be involved in juxtacrine signaling (49), the major effects of HB-EGF in the β-cell are likely mediated by the soluble form generated by proteolytic processing of proHB-EGF. In mesangial cells, glucose promotes HB-EGF shedding and EGFR transactivation through Src-dependent activation of metalloproteases (30). Our results showing that Src inhibition blocked glucose- but not HB-EGF–induced β-cell proliferation suggest that this phenomenon is also operative in β-cells. Consistent with this possibility, short-term exposure of MIN6 and human islets to glucose leads to phosphorylation of the Src family kinase YES (50). Overall, our data are consistent with the model proposed in Fig. 8, in which glucose promotes Src-dependent proHB-EGF processing, leading to HB-EGF shedding and stimulation of β-cell proliferation via paracrine and/or autocrine signaling through the EGFR.

mTOR is an essential mediator of mitogen-induced β-cell proliferation (51). Blocking mTOR activity prevents the mitogenic effects of glucose (16) and, as we showed in the current study, also mitigates HB-EGF–induced proliferation. Surprisingly, however, blocking HB-EGF had no effect on the increase in mTOR activity in response to glucose, yet HB-EGF inhibition completely prevented glucose-induced β-cell proliferation. Hence, we postulate the existence of a parallel signal emanating from EGFR acting alongside the mTOR pathway that is necessary for β-cell cycle engagement. A number of signaling effectors are known to act downstream of EGFR, including MAPK, PI3K/AKT (21), and IRS2 (52), that could contribute to the mitogenic response to HB-EGF (Fig. 8). Full characterization of the signaling pathway linking EGFR to the β-cell mitogenic response will require additional studies examining the potential implication of these kinases.

In conclusion, this study reveals a critical role of HB-EGF/EGFR signaling in glucose-induced β-cell proliferation in rat islets. Future studies will focus on further elucidating the underlying mechanism and assessing the importance of this pathway in human islet pathophysiology.

L.B.H. is currently affiliated with Regulatory and Quality Solutions, Cleveland, OH.

M.R.M. is currently affiliated with Syneos Health, Morrisville, NC.

Acknowledgments. The authors thank A. Levert (CRCHUM, Montreal, Quebec, Canada) for technical assistance with isolated experiments and R. Screaton (Sunnybrook Research Institute, Toronto, Ontario, Canada) for advice on human islet culture.

Funding. H.M. was supported by a doctoral studentship from the Fonds de Recherche du Québec-Santé. This study was supported by the National Institutes of Health (grants R01-DK-108905 to D.K.S. and R01-DK-58096 to V.P.) and the Canadian Institutes of Health Research (grant MOP 77686 to V.P.). V.P. holds the Canada Research Chair in Diabetes and Pancreatic Beta-Cell Function.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. H.M. and M.R.M. designed the experiments and acquired the data. H.M., M.R.M., D.K.S., J.G., and V.P. researched data, analyzed the results, and wrote the manuscript. All authors revised the manuscript and approved the final version. V.P. is the guarantor of this work and, as such, takes full responsibility for the work.

Prior Presentation. Parts of this study were presented at the 77th Scientific Sessions of the American Diabetes Association, San Diego, CA, 9–13 June 2017 and the 79th Scientific Sessions of the American Diabetes Association, San Francisco, CA, 7–11 June 2019.

1.
Kahn
SE
,
Cooper
ME
,
Del Prato
S
.
Pathophysiology and treatment of type 2 diabetes: perspectives on the past, present, and future
.
Lancet
2014
;
383
:
1068
1083
2.
Dor
Y
,
Brown
J
,
Martinez
OI
,
Melton
DA
.
Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation
.
Nature
2004
;
429
:
41
46
3.
Teta
M
,
Rankin
MM
,
Long
SY
,
Stein
GM
,
Kushner
JA
.
Growth and regeneration of adult beta cells does not involve specialized progenitors
.
Dev Cell
2007
;
12
:
817
826
4.
Okada
T
,
Liew
CW
,
Hu
J
, et al
.
Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance
.
Proc Natl Acad Sci U S A
2007
;
104
:
8977
8982
5.
Kim
H
,
Toyofuku
Y
,
Lynn
FC
, et al
.
Serotonin regulates pancreatic beta cell mass during pregnancy
.
Nat Med
2010
;
16
:
804
808
6.
Hakonen
E
,
Ustinov
J
,
Mathijs
I
, et al
.
Epidermal growth factor (EGF)-receptor signalling is needed for murine beta cell mass expansion in response to high-fat diet and pregnancy but not after pancreatic duct ligation
.
Diabetologia
2011
;
54
:
1735
1743
7.
El Ouaamari
A
,
Dirice
E
,
Gedeon
N
, et al
.
SerpinB1 promotes pancreatic β cell proliferation
.
Cell Metab
2016
;
23
:
194
205
8.
Moullé
VS
,
Vivot
K
,
Tremblay
C
,
Zarrouki
B
,
Ghislain
J
,
Poitout
V
.
Glucose and fatty acids synergistically and reversibly promote beta cell proliferation in rats
.
Diabetologia
2017
;
60
:
879
888
9.
Alonso
LC
,
Yokoe
T
,
Zhang
P
, et al
.
Glucose infusion in mice: a new model to induce beta-cell replication
.
Diabetes
2007
;
56
:
1792
1801
10.
Terauchi
Y
,
Takamoto
I
,
Kubota
N
, et al
.
Glucokinase and IRS-2 are required for compensatory beta cell hyperplasia in response to high-fat diet-induced insulin resistance
.
J Clin Invest
2007
;
117
:
246
257
11.
Porat
S
,
Weinberg-Corem
N
,
Tornovsky-Babaey
S
, et al
.
Control of pancreatic β cell regeneration by glucose metabolism
.
Cell Metab
2011
;
13
:
440
449
12.
Stamateris
RE
,
Sharma
RB
,
Hollern
DA
,
Alonso
LC
.
Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression
.
Am J Physiol Endocrinol Metab
2013
;
305
:
E149
E159
13.
Levitt
HE
,
Cyphert
TJ
,
Pascoe
JL
, et al
.
Glucose stimulates human beta cell replication in vivo in islets transplanted into NOD-severe combined immunodeficiency (SCID) mice
.
Diabetologia
2011
;
54
:
572
582
14.
Martinez
SC
,
Cras-Méneur
C
,
Bernal-Mizrachi
E
,
Permutt
MA
.
Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet β-cell
.
Diabetes
2006
;
55
:
1581
1591
15.
Kulkarni
RN
,
Brüning
JC
,
Winnay
JN
,
Postic
C
,
Magnuson
MA
,
Kahn
CR
.
Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes
.
Cell
1999
;
96
:
329
339
16.
Stamateris
RE
,
Sharma
RB
,
Kong
Y
, et al
.
Glucose induces mouse β-cell proliferation via IRS2, MTOR, and cyclin D2 but not the insulin receptor
.
Diabetes
2016
;
65
:
981
995
17.
Metukuri
MR
,
Zhang
P
,
Basantani
MK
, et al
.
ChREBP mediates glucose-stimulated pancreatic β-cell proliferation
.
Diabetes
2012
;
61
:
2004
2015
18.
Zhang
P
,
Kumar
A
,
Katz
LS
, et al
.
Induction of the ChREBPβ isoform is essential for glucose-stimulated β-cell proliferation
.
Diabetes
2015
;
64
:
4158
4170
19.
Stoeckman
AK
,
Ma
L
,
Towle
HC
.
Mlx is the functional heteromeric partner of the carbohydrate response element-binding protein in glucose regulation of lipogenic enzyme genes
.
J Biol Chem
2004
;
279
:
15662
15669
20.
Fontés
G
,
Zarrouki
B
,
Hagman
DK
, et al
.
Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass
.
Diabetologia
2010
;
53
:
2369
2379
21.
Zarrouki
B
,
Benterki
I
,
Fontés
G
, et al
.
Epidermal growth factor receptor signaling promotes pancreatic β-cell proliferation in response to nutrient excess in rats through mTOR and FOXM1
.
Diabetes
2014
;
63
:
982
993
22.
Song
Z
,
Fusco
J
,
Zimmerman
R
, et al
.
Epidermal growth factor receptor signaling regulates β cell proliferation in adult mice
.
J Biol Chem
2016
;
291
:
22630
22637
23.
Taylor
SR
,
Markesbery
MG
,
Harding
PA
.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and proteolytic processing by a disintegrin and metalloproteinases (ADAM): a regulator of several pathways
.
Semin Cell Dev Biol
2014
;
28
:
22
30
24.
Kelpe
CL
,
Johnson
LM
,
Poitout
V
.
Increasing triglyceride synthesis inhibits glucose-induced insulin secretion in isolated rat islets of Langerhans: a study using adenoviral expression of diacylglycerol acyltransferase
.
Endocrinology
2002
;
143
:
3326
3332
25.
Ferdaoussi
M
,
Bergeron
V
,
Zarrouki
B
, et al
.
G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1
.
Diabetologia
2012
;
55
:
2682
2692
26.
Hagman
DK
,
Latour
MG
,
Chakrabarti
SK
, et al
.
Cyclical and alternating infusions of glucose and intralipid in rats inhibit insulin gene expression and Pdx-1 binding in islets
.
Diabetes
2008
;
57
:
424
431
27.
Ghislain
J
,
Fontés
G
,
Tremblay
C
,
Kebede
MA
,
Poitout
V
.
Dual-reporter β-cell-specific male transgenic rats for the analysis of β-cell functional mass and enrichment by flow cytometry
.
Endocrinology
2016
;
157
:
1299
1306
28.
Schmidt
SF
,
Madsen
JG
,
Frafjord
KO
, et al
.
Integrative genomics outlines a biphasic glucose response and a ChREBP-RORγ axis regulating proliferation in β cells
.
Cell Rep
2016
;
16
:
2359
2372
29.
Hagège
H
,
Klous
P
,
Braem
C
, et al
.
Quantitative analysis of chromosome conformation capture assays (3C-qPCR)
.
Nat Protoc
2007
;
2
:
1722
1733
30.
Taniguchi
K
,
Xia
L
,
Goldberg
HJ
, et al
.
Inhibition of Src kinase blocks high glucose-induced EGFR transactivation and collagen synthesis in mesangial cells and prevents diabetic nephropathy in mice
.
Diabetes
2013
;
62
:
3874
3886
31.
Kozawa
J
,
Tokui
Y
,
Moriwaki
M
, et al
.
Regenerative and therapeutic effects of heparin-binding epidermal growth factor-like growth factor on diabetes by gene transduction through retrograde pancreatic duct injection of adenovirus vector
.
Pancreas
2005
;
31
:
32
42
32.
Means
AL
,
Ray
KC
,
Singh
AB
, et al
.
Overexpression of heparin-binding EGF-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia
.
Gastroenterology
2003
;
124
:
1020
1036
33.
Tokui
Y
,
Kozawa
J
,
Yamagata
K
, et al
.
Neogenesis and proliferation of beta-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector
.
Biochem Biophys Res Commun
2006
;
350
:
987
993
34.
Rescan
C
,
Le Bras
S
,
Lefebvre
VH
, et al
.
EGF-induced proliferation of adult human pancreatic duct cells is mediated by the MEK/ERK cascade
.
Lab Invest
2005
;
85
:
65
74
35.
Suarez-Pinzon
WL
,
Lakey
JR
,
Brand
SJ
,
Rabinovitch
A
.
Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet beta-cells from pancreatic duct cells and an increase in functional beta-cell mass
.
J Clin Endocrinol Metab
2005
;
90
:
3401
3409
36.
Castillo
GM
,
Nishimoto-Ashfield
A
,
Banerjee
AA
,
Landolfi
JA
,
Lyubimov
AV
,
Bolotin
EM
.
Omeprazole and PGC-formulated heparin binding epidermal growth factor normalizes fasting blood glucose and suppresses insulitis in multiple low dose streptozotocin diabetes model
.
Pharm Res
2013
;
30
:
2843
2854
37.
Elenius
K
,
Paul
S
,
Allison
G
,
Sun
J
,
Klagsbrun
M
.
Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation
.
EMBO J
1997
;
16
:
1268
1278
38.
DiGruccio
MR
,
Mawla
AM
,
Donaldson
CJ
, et al
.
Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets
.
Mol Metab
2016
;
5
:
449
458
39.
Hakonen
E
,
Ustinov
J
,
Palgi
J
,
Miettinen
PJ
,
Otonkoski
T
.
EGFR signaling promotes β-cell proliferation and survivin expression during pregnancy
.
PLoS One
2014
;
9
:
e93651
40.
Buteau
J
,
Foisy
S
,
Joly
E
,
Prentki
M
.
Glucagon-like peptide 1 induces pancreatic beta-cell proliferation via transactivation of the epidermal growth factor receptor
.
Diabetes
2003
;
52
:
124
132
41.
Huotari
MA
,
Palgi
J
,
Otonkoski
T
.
Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel beta-cell mitogen
.
Endocrinology
1998
;
139
:
1494
1499
42.
Li
L
,
Seno
M
,
Yamada
H
,
Kojima
I
.
Promotion of beta-cell regeneration by betacellulin in ninety percent-pancreatectomized rats
.
Endocrinology
2001
;
142
:
5379
5385
43.
Kuntz
E
,
Broca
C
,
Komurasaki
T
, et al
.
Effect of epiregulin on pancreatic beta cell growth and insulin secretion
.
Growth Factors
2005
;
23
:
285
293
44.
Krakowski
ML
,
Kritzik
MR
,
Jones
EM
, et al
.
Transgenic expression of epidermal growth factor and keratinocyte growth factor in beta-cells results in substantial morphological changes
.
J Endocrinol
1999
;
162
:
167
175
45.
Huotari
MA
,
Miettinen
PJ
,
Palgi
J
, et al
.
ErbB signaling regulates lineage determination of developing pancreatic islet cells in embryonic organ culture
.
Endocrinology
2002
;
143
:
4437
4446
46.
Li
M
,
Miyagawa
J
,
Moriwaki
M
, et al
.
Analysis of expression profiles of islet-associated transcription and growth factors during beta-cell neogenesis from duct cells in partially duct-ligated mice
.
Pancreas
2003
;
27
:
345
355
47.
Tu
Z
,
Keller
MP
,
Zhang
C
, et al
.
Integrative analysis of a cross-loci regulation network identifies App as a gene regulating insulin secretion from pancreatic islets
.
PLoS Genet
2012
;
8
:
e1003107
48.
Bensellam
M
,
Van Lommel
L
,
Overbergh
L
,
Schuit
FC
,
Jonas
JC
.
Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations
.
Diabetologia
2009
;
52
:
463
476
49.
Ray
KC
,
Blaine
SA
,
Washington
MK
, et al
.
Transmembrane and soluble isoforms of heparin-binding epidermal growth factor-like growth factor regulate distinct processes in the pancreas
.
Gastroenterology
2009
;
137
:
1785
1794
50.
Yoder
SM
,
Dineen
SL
,
Wang
Z
,
Thurmond
DC
.
YES, a Src family kinase, is a proximal glucose-specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet β cells
.
J Biol Chem
2014
;
289
:
11476
11487
51.
Balcazar
N
,
Sathyamurthy
A
,
Elghazi
L
, et al
.
mTORC1 activation regulates beta-cell mass and proliferation by modulation of cyclin D2 synthesis and stability
.
J Biol Chem
2009
;
284
:
7832
7842
52.
Oh
YS
,
Shin
S
,
Lee
YJ
,
Kim
EH
,
Jun
HS
.
Betacellulin-induced beta cell proliferation and regeneration is mediated by activation of ErbB-1 and ErbB-2 receptors
.
PLoS One
2011
;
6
:
e23894
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at https://www.diabetesjournals.org/content/license.

Supplementary data