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. 2013 Feb 5;154(3):1039–1046. doi: 10.1210/en.2012-1923

Glucocorticoid-Induced Suppression of β-Cell Proliferation Is Mediated by Mig6

E Scott Colvin 1,*, Hong-Yun Ma 1,*, Yi-Chun Chen 1, Angelina M Hernandez 1, Patrick T Fueger 1,✉
PMCID: PMC3578994  PMID: 23384834

Abstract

Glucocorticoids can cause steroid-induced diabetes or accelerate the progression to diabetes by creating systemic insulin resistance and decreasing functional β-cell mass, which is influenced by changes in β-cell function, growth, and death. The synthetic glucocorticoid agonist dexamethasone (Dex) is deleterious to functional β-cell mass by decreasing β-cell function, survival, and proliferation. However, the mechanism by which Dex decreases β-cell proliferation is unknown. Interestingly, Dex induces the transcription of an antiproliferative factor and negative regulator of epidermal growth factor receptor signaling, Mig6 (also known as gene 33, RALT, and Errfi1). We, therefore, hypothesized that Dex impairs β-cell proliferation by increasing the expression of Mig6 and thereby decreasing downstream signaling of epidermal growth factor receptor. We found that Dex induced Mig6 and decreased [3H]thymidine incorporation, an index of cellular replication, in mouse, rat, and human islets. Using adenovirally delivered small interfering RNA targeted to Mig6 in rat islets, we were able to limit the induction of Mig6 upon exposure to Dex, compared with islets treated with a control virus, and completely rescued the Dex-mediated impairment in replication. We demonstrated that both Dex and overexpression of Mig6 attenuated the phosphorylation of ERK1/2 and blocked the G1/S transition of the cell cycle. In conclusion, Mig6 functions as a molecular brake for β-cell proliferation during glucocorticoid treatment in β-cells, and thus, Mig6 may be a novel target for preventing glucocorticoid-induced impairments in functional β-cell mass.

Glucose homeostasis is primarily maintained by the intricate balance between insulin, which stimulates glucose disposal and suppresses hepatic glucose production, and the counter-regulatory hormones, which oppose the actions of insulin. The metabolic demand for insulin is tightly coupled to the functional β-cell mass, which is dependent on the number and size of β-cells and their capacity to secrete insulin (1, 2). When the metabolic demand for insulin rises, such as during pregnancy (3) or insulin resistance (4), so too does the functional β-cell mass.

As potent counter-regulatory hormones, glucocorticoids induce insulin resistance and can cause steroid-induced diabetes or accelerate the progression from prediabetes to frank diabetes (5, 6). The normal compensatory response to systemic insulin resistance is to increase functional β-cell mass by enhancing β-cell function and/or increasing the number of β-cells (7). Thus, steroid-induced diabetes occurs when the functional β-cell mass cannot appropriately adapt to the demand placed on the β-cells by the existing insulin resistance. Whereas glucocorticoids markedly suppress insulin secretion by altering the expression of the transcription factors FoxO1 and Pdx-1 in the pancreatic β-cell (8), little is known regarding how they impair β-cell proliferation.

Mitogen-inducible gene 6 (Mig6; also called gene 33, receptor-associated late transducer [ralt], and ErbB receptor feedback inhibitor 1 [errfi1]) was reported to be induced by glucocorticoid receptor (GR) activation decades ago (9), yet the function of Mig6 has only become revealed in the last decade (10). Mig6 serves as an endogenous feedback inhibitor of epidermal growth factor (EGF) receptor (EGFR) or hepatic growth factor (HGF)/Met signaling (10–13) and thus functions as a molecular brake for proproliferative pathways. A loss of Mig6 has been reported to increase proliferation in hepatocytes (14) and increase the development of tumors in various mouse models (13, 15). Therefore, we hypothesized that the induction of Mig6 mediates glucocorticoid-induced impairments in β-cell proliferation.

We report here that Mig6 is induced in rodent and human islets after exposure to the synthetic glucocorticoid dexamethasone (Dex) and is associated with impairments in β-cell replication. Mig6, like Dex, blocks the G1/S transition of the cell cycle and mediates the suppression of islet replication after exposure to glucocorticoids. Thus, suppressing the induction or actions of Mig6 could be warranted for abrogating steroid-induced diabetes.

Materials and Methods

Cell culture and rodent islet isolation

INS-1-derived 832/13 rat insulinoma cells were cultured as described previously (17). Pancreatic islets were harvested from male Wistar rats weighing approximately 250 g (18, 19) and 8-wk-old C57Bl/6J mice (20, 21), under a protocol approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.

Measurement of mRNA levels

RNA was harvested from 832/13 cells or 20-50 islets using the RNeasy mini or micro kits (QIAGEN, Valencia, California), respectively. First-strand cDNA synthesis was performed with 0.5 μg RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, California). Real-time PCR was performed using an Eppendorf realplex2 detection system and software and Taqman Gene Expression Master Mix reagents with Taqman Gene Expression assays (Applied Biosystems). The threshold cycle (CT) method (22) was used to calculate relative quantities of mRNA products in each sample, and all samples were normalized for total cDNA by normalizing to the CT value of the control glyceraldehyde 3-phosphate dehydrogenase.

Immunoblot analysis

Whole-cell lysates were prepared using radioimmunoprecipitation assay buffer supplemented with protease (Santa Cruz Biotechnology, Santa Cruz, California) and phosphatase inhibitor cocktails (Sigma Chemical Co, St Louis, Missouri). Protein samples (30 μg) were resolved on 10% Bis-Tris-HCl-buffered polyacrylamide gels (Invitrogen, Carlsbad, California) and transferred to polyvinylidene difluoride membranes. Membranes were blocked using a SNAP i.d. apparatus (Millipore Corp, Bedford, Massachusetts). Primary antibodies were diluted in polyvinylpyrrolidone (23) as follows: anti-Mig6 (1:1000; Cell Signaling Technology, Beverly, Massachusetts; catalog no. 2440), antiphosphorylated Akt (1:1000; Cell Signaling Technology; catalog no. 4056), anti-Akt (1:1000; Cell Signaling Technology; catalog no. 2920), antiphosphorylated ERK1/2 (1:1000; Cell Signaling Technology; catalog no. 9101), anti-ERK1/2 (1:1000; Cell Signaling; catalog no.4696), or antiactin (1:5000 as a loading control; MP Biomedicals; Irvine, California; catalog no. 691001). Membranes were incubated overnight at 4°C, and primary antibodies were detected using fluorophore-labeled secondary antibodies (IRDye 800 and IRDye 700) and visualized using the LiCor Odyssey system (LI-COR Biosciences, Lincoln, Nebraska).

Apoptosis assays

To measure apoptotic cell death, caspase 3 cleavage and activity were detected by immunoblotting and enzymatic assay, respectively, on whole-cell lysates without protease inhibitors. Procedures for immunoblotting were followed as above using an antibody to caspase 3 (1:1000, no. 9662; Cell Signaling Technology). Caspase 3 enzymatic activity was assayed with the Caspase 3 Fluorometric Assay Kit (Upstate Biotechnology, Inc., Lake Placid, New York) and a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, California). Protein concentration was measured using the BCA Protein Assay Kit (Pierce Chemical Co, Rockford, Illinois) to normalize caspase 3 activity.

[3H]Thymidine incorporation

DNA synthesis rates were measured by measurement of incorporation of [3H]methylthymidine into genomic DNA of 832/13 cells and isolated islets as described previously (24–26). Briefly, [3H]methylthymidine was added at a final concentration of 1 μCi/ml to 832/13 cells in 12-well plates during the last 4 h of cell culture or to pools of 100-200 islets during the last 18 h of cell culture. The 832/13 cells were washed three times with cold phosphate buffered saline. DNA was precipitated with two 500-μl aliquots of cold 10% trichloroacetic acid and solubilized by addition of 250 μl 0.3 N NaOH. Groups of 20 islets were picked in triplicate, washed, and centrifuged twice at 300 × g for 3 min at 4°C. DNA was precipitated with 500 μl cold 10% trichloroacetic acid and solubilized by addition of 80 μl 0.3 N NaOH. The amount of [3H]thymidine incorporated into DNA was measured by liquid scintillation counting and normalized to total cellular protein.

Human islet experiments

Human islets were obtained from β-Pro, LLC (Charlottesville, Virginia). Islet preparations were cultured and used for measurements of [3H]thymidine incorporation exactly as described for rodent islet cultures.

Use of recombinant adenoviruses

For gene overexpression studies, recombinant adenoviruses containing the rat Mig6 cDNA (AdCMV-Mig6; kindly provided by from Drs. Xu and Kyriakis) or the green fluorescent protein (GFP) gene (AdCMV-GFP) were prepared (27) and used (28, 29) as previously described. For gene suppression studies, adenoviruses containing small interfering RNAs (siRNAs) specific to rat Mig6 (Ad-siMig6) or with no known gene homology (Ad-siControl) were prepared and used as described previously (29, 30). Sequences for the siRNAs are available upon request from the authors. Primary rat islets and 832/13 cells were treated cultured in the presence of adenoviruses for 16 h and then cultured in virus-free media for the remainder of the experiments.

Cell cycle analysis

832/13 cells were treated with dimethylsulfoxide (DMSO) or Dex or transduced with AdCMV-GFP or AdCMV-Mig6 and cultured overnight. For cell cycle analysis, cells were labeled with propidium iodide using the Guava Cell Cycle Reagent (EMD Millipore, Billerica, Massachusetts), and cellular DNA content was analyzed using flow cytometry.

Statistical methods

Student's t test or ANOVA was used to detect statistical differences (P < .05). Differences within ANOVA were determined using Tukey's post hoc tests. All data are reported as means ± SEM.

Results

GR activation stimulates Mig6 expression, which inhibits β-cell proliferation

Stress hormones such as glucocorticoids can lead to deleterious effects on pancreatic islets, including decreased β-cell proliferation (31). We thus measured the expression of Mig6 in INS-1-derived 832/13 cells and β-cell replication after treatment with Dex. We demonstrate that Dex induced Mig6 and inhibited β-cell replication in a dose-dependent manner, as indexed by [3H]thymidine incorporation, in 832/13 cells (Figure 1). Because the effect of Dex on Mig6 expression and [3H]thymidine incorporation began to plateau at a concentration of 100 nM, we used this dose in 832/13 cells for the remainder of the study and report a near doubling in Mig6 protein content (Figure 2). Importantly, overnight exposure to Dex both decreased [3H]thymidine incorporation and induced the expression of Mig6 in mouse, rat, and human islets (Figure 3).

Figure 1.

Figure 1.

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Dexamethasone Increases Mig6 Expression and Decreases β-Cell Proliferation. 832/13 cells were treated with vehicle or increasing doses of Dex. Mig6 mRNA (A) and protein (B) were measured, and cell proliferation (C) was assessed by [3H]thymidine incorporation after 18 hours. Data are normalized to DMSO-treated cells and are presented as means ± SEM; n = 5. *P < .05 vs 0; #P < .05 vs 0 and 10.

Figure 2.

Figure 2.

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Dexamethasone Increases Mig6 Protein Content. 832/13 cells were treated with vehicle (DMSO) or Dex (100 nM) for 18 hours. Mig6 protein content was assessed using immunoblotting; shown are a representative blot (A) and quantified results (B). Data are normalized to DMSO-treated cells and are presented as means ± SEM; n = 3. *P < .05 vs DMSO.

Figure 3.

Figure 3.

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Dexamethasone Decreases Islet Proliferation and Increases Mig6 Expression Rat, mouse, and human islets were treated with DMSO or 1 mM Dex for 48 hours. Mig-6 mRNA expression was quantified by RT-PCR (A), and proliferation was measured by [3H]thymidine incorporation (B). Data are normalized to DMSO-treated islets and presented as means ± SEM; n = 3. *P < .05 vs DMSO.

The decreased [3H]thymidine incorporation with Dex treatment is not likely due to increased cell death under the experimental conditions used. Dex did not increase the abundance of cleaved caspase 3 or increase caspase 3 activity (a hallmark of apoptosis) in 832/13 cells, whereas the endoplasmic reticulum stress inducer thapsigargin induced apoptosis (Supplemental Figure 1 published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org).

To determine whether Mig6 alone has the ability to regulate islet proliferation, we overexpressed Mig6 in isolated rat islets using a recombinant adenovirus (AdCMV-Mig6) and compared them with islets treated with a control adenovirus overexpressing green fluorescent protein (AdCMV-GFP). AdCMV-Mig6-transduced rat islets had decreased [3H]thymidine incorporation compared with control AdCMV-GFP-transduced islets (Figure 4).

Figure 4.

Figure 4.

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Mig6 Suppresses Islet Proliferation. Isolated rat islets were transduced with control or Mig6-overexpressing adenoviruses (AdCMV-GFP or AdCMV-Mig6, respectively), overexpression was confirmed by immunoblotting (A), and islet proliferation was assessed by [3H]thymidine incorporation after 48 hours (B). Data are presented as means ± SEM; n = 3. *P < .05 vs AdCMV-GFP.

To identify which phase of the cell cycle is blocked by glucocorticoids and Mig6, we performed flow cytometry analysis in 832/13 cells (Figure 5). Dex treatment, compared with DMSO-treated controls, led to a decrease in the percentage of cells in the S and G2/M phases and commensurate rise in the percentage of cells in G0/G1, thereby suggesting a block in at least the G1/S transition. Likewise, Mig6 overexpression also decreased the cells in S phase and increased those in G0/G1, again suggesting a block in the G1/S transition.

Figure 5.

Figure 5.

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Dexamethasone and Mig6 Block the G1/S Transition. 832/13 cells were treated with DMSO or 100 nM Dex for 24 hours (A) or transduced with control or Mig6-overexpressing adenoviruses (AdCMV-GFP or AdCMV-Mig6, respectively) for 24 hours (B). Phases of the cell cycle were assessed using flow cytometry. Data are expressed as a percentage of the total cell population and are presented as means ± SEM; n = 3. *P < .05 vs DMSO or AdCMV-GFP.

GR activation and Mig6 suppress proliferation by attenuating ERK1/2 phosphorylation

Because Dex has been previously reported to reduce Akt phosphorylation in rat RINm5F insulinoma cells (32) and fibroblasts (10) and Akt is able to stimulate β-cell proliferation (33), we sought to determine whether Dex exposure and Mig6 overexpression could also decrease Akt phosphorylation in 832/13 cells. Culturing 832/13 cells in Dex overnight decreased Akt phosphorylation compared with control DMSO-treated cells (Figure 6). However, Mig6 overexpression was not able to prevent Akt phosphorylation after stimulation with serum (Supplemental Figure 2).

Figure 6.

Figure 6.

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Dexamethasone Suppresses Akt and ERK Activation. 832/13 cells were treated with DMSO or 100 nM Dex for 24 hours. Total and phosphorylated Akt and ERK were assessed by immunoblotting; actin served as a loading control. Shown are representative immunoblots (A and B) and quantified results (C and D). Data are presented as means ± SEM; n = 3. *P < .05 vs. DMSO.

Because glucocorticoids have also been shown to attenuate ERK1/2 phosphorylation (34, 35), we assessed whether Dex exposure and Mig6 overexpression could also decrease ERK1/2 phosphorylation in 832/13 cells. Similar to Akt, overnight treatment with Dex decreased ERK1/2 phosphorylation (Figure 6). Mig6 overexpression tended to attenuate ERK1/2 phosphorylation after stimulation with serum (Supplemental Figure 2), and thus we focused on ERK1/2, and not Akt, phosphorylation.

To determine the impact of Dex and Mig6 on EGFR-mediated ERK1/2 phosphorylation in β-cells, we performed both overexpression and siRNA-mediated knockdown experiments in 832/13 cells cultured in the presence and absence of Dex (Figure 7). After a brief starvation (4 h), cells were stimulated with EGF (10 ng/ml for 5 min). Overexpression and knockdown of Mig6 were confirmed by immunoblotting and RT-PCR; the knockdown efficiency was approximately 60% (1.00 ± 0.19 vs 0.43 ± 0.11 relative Mig6 expression for Ad-siControl- and Ad-siMig6-treated cells, respectively). Whereas Mig6 overexpression suppressed EGF-stimulated ERK1/2 phosphorylation, siRNA-mediated suppression of Mig6 enhanced it. EGF-stimulated ERK1/2 phosphorylation was also attenuated by Dex, and it was further reduced with Mig6 overexpression. In contrast, siRNA-mediated suppression of Mig6 rescued the Dex-mediated impairment in EGF-stimulated ERK1/2 phosphorylation.

Figure 7.

Figure 7.

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Mig6 Regulates EGF-Stimulated ERK Activation. 832/13 cells were transduced with control or Mig6-overexpressing adenoviruses (AdCMV-GFP or AdCMV-Mig6, respectively [A and B] for up to 48 hours or adenoviruses expressing a scrambled control siRNA or one targeting Mig6 (Ad-siControl or Ad-siMig6, respectively [C and D] for up to 72 hours). In the final 18 hours, cells were exposed to DMSO or Dex (100 nM). Cells were starved for 4 h and then exposed to EGF for 5 minutes. Total and phosphorylated ERK were assessed by immunoblotting; actin served as a loading control. Shown are representative immunoblots (A and C) and quantified results (B and D). Data are presented as means ± SEM; n = 3. *P < .05 vs AdCMV-GFP or Ad-siControl; #P < .05 vs all other EGF-stimulated groups in panel B or Ad-siControl + Dex in panel D.

Attenuation of Mig6 induction abrogates Dex-mediated impairments in islet proliferation

To determine whether the rise in Mig6 contributes to the impairment in islet replication after Dex treatment, we constructed a siRNA-expressing adenovirus (Ad-siMig6) to suppress the expression of Mig6. Mig6 expression was reduced to 49 ± 4% in rat islets transduced with Ad-siMig6 compared with Ad-siControl (Figure 8A). Upon treatment with Dex, Ad-siMig6-treated rat islets had attenuated Mig6 expression compared with Ad-siControl-treated islets. Importantly, the suppression of Mig6 in rat islets prevented the Dex-induced impairment in [3H]thymidine incorporation (Figure 8B).

Figure 8.

Figure 8.

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Attenuation of Mig6 Induction Ameliorates Dex-Mediated Impairments in Islet Proliferation. Rat islets were transduced with Ad-siControl or Ad-siMig6 and treated with DMSO or 1 mM Dex for 24 hours. Mig6 mRNA expression was quantified by RT-PCR (A), and proliferation was measured by [3H]thymidine incorporation (B). Data are normalized to DMSO- and Ad-siControl-treated islets and presented as means ± SEM; n = 5. *P < .05 vs DMSO; #P < .05 vs Ad-siControl.

Discussion

Steroid-induced diabetes is one of the unwanted side effects opposing the beneficial actions of glucocorticoid treatment (36, 37). Whereas glucocorticoids can provoke skeletal muscle and hepatic insulin resistance (38–40), they can also trigger deleterious effects on pancreatic islets, including decreased β-cell function (8, 31, 41), survival (31, 42), and proliferation (31). In fact, the glucocorticoid-free immunosuppressive regimen was one of the significant advances with the Edmonton Protocol for islet transplantation (43). Thus, defining the mechanisms responsible for the deleterious effects of glucocorticoids on pancreatic islets is essential for preventing diabetes in cases where the use of glucocorticoids may be required such as during organ or islet transplantation or trauma.

Several decades ago, Messina (9) demonstrated that the synthetic GR agonist Dex stimulated the expression of Mig6 in rat hepatoma cells. However, for almost 30 yr the function of Mig6 remained unknown until the demonstration that Mig6 is an endogenous inhibitor of EGFR (10). A similar function of Mig6 in HGF-Met signaling has also been established (11). Hence, we postulated that the induction of Mig6 and resulting feedback inhibition of EGFR or HGF/Met signaling would provide the mechanism for how Dex inhibits β-cell proliferation. We demonstrate here that Dex, in a dose-dependent manner, induces Mig6 and inhibits β-cell replication in a β-cell line and that overnight exposure to Dex decreases replication in mouse, rat, and human islets while, at the same time, inducing the expression of Mig6. Importantly, attenuating the Dex-mediated induction of Mig6 rescued rat islets from impairments in replication. Thus, suppressing Mig6 or inhibiting its function may be useful for preventing steroid-induced diabetes.

Mig6 is a multiadapter protein that, along with its ability to bind to EGFR, contains a proline-rich region for binding to SH3 domains, thereby permitting it to interact with molecules such as Grb2 and phosphatidylinositol 3-kinase, and a cdc42/Rac interaction and binding domain for interactions with cdc42. Mig6 has been largely thought to function by binding to EGFR family members (and other tyrosine kinase receptors) to prevent trans-autophosphorylation and target the receptor for degradation through the endosomes and lysosomes (44, 45). Nevertheless, Mig6 has the potential to impact a cadre of signaling pathways related to the growth/replication and function of the cell.

Both Dex and Mig6 overexpression blocked the cell cycle at the G1/S transition. The mechanism whereby Dex inhibits DNA synthesis and replication in β-cells could possibly involve a down-regulation of Akt and/or ERK signaling because others have established that Dex suppresses these pathways in hepatocytes (46). Indeed, we demonstrated a substantial reduction in phosphorylated Akt and ERK1/2 after treatment with Dex. However, as Mig6 overexpression is only able to block ERK and not Akt signaling in response to EGF stimulation in the results presented here, we would suggest that it is Dex's suppression of ERK signaling that is responsible for the inhibition of proliferation in β-cells. A similar mechanism has been proposed for the suppression of β-cell proliferation by proinflammatory cytokines (47), and ERK signaling has clearly been established to regulate the progression through the G1 phase and entry into the S phase of the cell cycle (48, 49). Thus, activating ERK signaling may prove to be useful in abolishing the glucocorticoid-induced impairments in β-cell replication.

The ability of Mig6 to block cell cycle progression in β-cells is not unexpected given its role in regulating proliferation in other cell types. Using tissue-specific knockout mice, several groups have reported an increase in the oncogenic or growth potential in cells lacking Mig6 (11, 13, 14, 50). Thus, whereas Mig6 may be a novel therapeutic target for counteracting the deleterious effects of glucocorticoids on functional β-cell mass, caution must be used in the design and implementation of agents targeting Mig6 in order to prevent the development of cancers.

Agents such as glucocorticoids often target multiple processes controlling functional β-cell mass. As mentioned above, glucocorticoids have been reported to decrease β-cell function (8, 31, 41), survival (31, 42), and proliferation (31). The induction of Mig6 may help to explain how glucocorticoids impact functional β-cell mass on multiple levels. We report here that Mig6 mediates the glucocorticoid-induced impairments in β-cell proliferation. Whereas Dex did not induce apoptosis in the experimental conditions used in the present study, we have recently demonstrated that Mig6 also antagonizes β-cell survival and promotes β-cell death (16). It remains to be determined whether Mig6 also impacts β-cell function. Nevertheless, Mig6 is emerging as a multiregulator of functional β-cell mass.

In summary, glucocorticoid treatment induces a potent anti-inflammatory response but comes at the expense of impaired glucose homeostasis through the development insulin resistance and decreased functional β-cell mass. Glucocorticoids can robustly induce Mig6 in the β-cell, which attenuates ERK signaling, thereby suppressing cellular replication via disruption of the G1/S transition. Thus, abrogating the induction of Mig6 or preventing its actions on ERK signaling may be useful for preventing impairments in functional β-cell mass associated with glucocorticoid treatment.

Supplementary Material

Supplemental Data
supp_154_3_1039__index.html (827B, html)

Acknowledgments

We thank Drs. Dazhong Xu and John Kyriakis for the Mig6-overexpressing adenovirus and Natalie Stull for expert technical assistance with islet isolation.

This work was supported by National Institutes of Health Grant DK078732 (to P.T.F.) and a Showalter Research Trust Award from Indiana University School of Medicine (to P.T.F.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
Dex
dexamethasone
DMSO
dimethylsulfoxide
EGF
epidermal growth factor
EGFR
EGF receptor
GFP
green fluorescent protein
GR
glucocorticoid receptor
HGF
hepatic growth factor
siRNA
small interfering RNA.

References

  • 1. Bouwens L, Rooman I. Regulation of pancreatic β-cell mass. Physiol Rev. 2005;85:1255–1270 [DOI] [PubMed] [Google Scholar]
  • 2. Finegood DT, Scaglia L, Bonner-Weir S. Dynamics of β-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes. 1995;44:249–256 [DOI] [PubMed] [Google Scholar]
  • 3. Rieck S, Kaestner KH. Expansion of β-cell mass in response to pregnancy. Trends Endocrinol Metab. 2010;21:151–158 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Sachdeva MM, Stoffers DA. Minireview: meeting the demand for insulin: molecular mechanisms of adaptive postnatal β-cell mass expansion. Mol Endocrinol. 2009;23:747–758 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Miller SE, Neilson JM. Clinical features of the diabetic syndrome appearing after steroid therapy. Postgrad Med J. 1964;40:660–669 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Volgi JR, Baldwin D., Jr Glucocorticoid therapy and diabetes management. Nurs Clin North Am. 2001;36:333–339, viii [PubMed] [Google Scholar]
  • 7. Weir GC, Bonner-Weir S. Five stages of evolving β-cell dysfunction during progression to diabetes. Diabetes 53 Suppl. 2004;3:S16–S21 [DOI] [PubMed] [Google Scholar]
  • 8. Chen F, Zhu Y, Tang X, Sun Y, Jia W, Han X. Dynamic regulation of PDX-1 and FoxO1 expression by FoxA2 in dexamethasone-induced pancreatic β-cells dysfunction. Endocrinology. 2011;152:1779–1788 [DOI] [PubMed] [Google Scholar]
  • 9. Messina JL. Insulin and dexamethasone regulation of a rat hepatoma messenger ribonucleic acid: insulin has a transcriptional and a posttranscriptional effect. Endocrinology. 1989;124:754–761 [DOI] [PubMed] [Google Scholar]
  • 10. Xu D, Makkinje A, Kyriakis JM. Gene 33 is an endogenous inhibitor of epidermal growth factor (EGF) receptor signaling and mediates dexamethasone-induced suppression of EGF function. J Biol Chem. 2005;280:2924–2933 [DOI] [PubMed] [Google Scholar]
  • 11. Pante G, Thompson J, Lamballe F, et al. Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth. J Cell Biol. 2005;171:337–348 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Zhang X, Pickin KA, Bose R, Jura N, Cole PA, Kuriyan J. Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface. Nature. 2007;450:741–744 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Ferby I, Reschke M, Kudlacek O, et al. Mig6 is a negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation. Nat Med. 2006;12:568–573 [DOI] [PubMed] [Google Scholar]
  • 14. Reschke M, Ferby I, Stepniak E, et al. Mitogen-inducible gene-6 is a negative regulator of epidermal growth factor receptor signaling in hepatocytes and human hepatocellular carcinoma. Hepatology. 2010;51:1383–1390 [DOI] [PubMed] [Google Scholar]
  • 15. Zhang YW, Staal B, Su Y, et al. Evidence that MIG-6 is a tumor-suppressor gene. Oncogene. 2007;26:269–276 [DOI] [PubMed] [Google Scholar]
  • 16. Chen YC, Colvin ES, Maier BF, Mirmira RG, Fueger PT. Mitogen-inducible gene 6 triggers apoptosis and exacerbates ER stress-induced β-cell death. Mol Endocrinol. 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, Newgard CB. Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes. 2000;49:424–430 [DOI] [PubMed] [Google Scholar]
  • 18. Naber SP, McDonald JM, Jarett L, McDaniel ML, Ludvigsen CW, Lacy PE. Preliminary characterization of calcium binding in islet-cell plasma membranes. Diabetologia. 1980;19:439–444 [DOI] [PubMed] [Google Scholar]
  • 19. Milburn JL, Jr., Hirose H, Lee YH, et al. Pancreatic β-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. J Biol Chem. 1995;270:1295–1299 [DOI] [PubMed] [Google Scholar]
  • 20. Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ. A protocol for islet isolation from mouse pancreas. Nat Protoc. 2009;4:1649–1652 [DOI] [PubMed] [Google Scholar]
  • 21. Yang Z, Chen M, Fialkow LB, Ellett JD, Wu R, Nadler JL. The novel anti-inflammatory compound, lisofylline, prevents diabetes in multiple low-dose streptozotocin-treated mice. Pancreas. 2003;26:e99–e104 [DOI] [PubMed] [Google Scholar]
  • 22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Δ Δ C(T)) method. Methods. 2001;25:402–408 [DOI] [PubMed] [Google Scholar]
  • 23. Haycock JW. Polyvinylpyrrolidone as a blocking agent in immunochemical studies. Anal Biochem. 1993;208:397–399 [DOI] [PubMed] [Google Scholar]
  • 24. Fueger PT, Hernandez AM, Chen YC, Colvin ES. 2012 Assessing replication and β-cell function in adenovirally-transduced isolated rodent islets. J Vis Exp. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Schisler JC, Fueger PT, Babu DA, et al. Stimulation of human and rat islet β-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1. Mol Cell Biol. 2008;28:3465–3476 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Fueger PT, Schisler JC, Lu D, et al. Trefoil factor 3 stimulates human and rodent pancreatic islet β-cell replication with retention of function. Mol Endocrinol. 2008;22:1251–1259 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998;95:2509–2514 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Becker TC, Noel RJ, Coats WS, et al. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 1994;43 Pt A:161–189 [DOI] [PubMed] [Google Scholar]
  • 29. Schisler JC, Jensen PB, Taylor DG, et al. The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet β cells. Proc Natl Acad Sci USA. 2005;102:7297–7302 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Bain JR, Schisler JC, Takeuchi K, Newgard CB, Becker TC. An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans. Diabetes. 2004;53:2190–2194 [DOI] [PubMed] [Google Scholar]
  • 31. Weinhaus AJ, Bhagroo NV, Brelje TC, Sorenson RL. Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy. Endocrinology. 2000;141:1384–1393 [DOI] [PubMed] [Google Scholar]
  • 32. Zhang X, Yong W, Lv J, et al. Inhibition of forkhead box O1 protects pancreatic β-cells against dexamethasone-induced dysfunction. Endocrinology. 2009;150:4065–4073 [DOI] [PubMed] [Google Scholar]
  • 33. Fatrai S, Elghazi L, Balcazar N, et al. Akt induces β-cell proliferation by regulating cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 activity. Diabetes. 2006;55:318–325 [DOI] [PubMed] [Google Scholar]
  • 34. Engelbrecht Y, de Wet H, Horsch K, Langeveldt CR, Hough FS, Hulley PA. Glucocorticoids induce rapid up-regulation of mitogen-activated protein kinase phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase and impair proliferation in human and mouse osteoblast cell lines. Endocrinology. 2003;144:412–422 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Greenberg AK, Hu J, Basu S, et al. Glucocorticoids inhibit lung cancer cell growth through both the extracellular signal-related kinase pathway and cell cycle regulators. Am J Respir Cell Mol Biol. 2002;27:320–328 [DOI] [PubMed] [Google Scholar]
  • 36. Hoogwerf B, Danese RD. Drug selection and the management of corticosteroid-related diabetes mellitus. Rheum Dis Clin North Am. 1999;25:489–505 [DOI] [PubMed] [Google Scholar]
  • 37. Schacke H, Docke WD, Asadullah K. Mechanisms involved in the side effects of glucocorticoids. Pharmacol Ther. 2002;96:23–43 [DOI] [PubMed] [Google Scholar]
  • 38. Dimitriadis G, Leighton B, Parry-Billings M, et al. Effects of glucocorticoid excess on the sensitivity of glucose transport and metabolism to insulin in rat skeletal muscle. Biochem J. 1997;321(3):707–712 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Tounian P, Schneiter P, Henry S, Delarue J, Tappy L. Effects of dexamethasone on hepatic glucose production and fructose metabolism in healthy humans. Am J Physiol. 1997;273:E315–E320 [DOI] [PubMed] [Google Scholar]
  • 40. Ruzzin J, Wagman AS, Jensen J. Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor. Diabetologia. 2005;48:2119–2130 [DOI] [PubMed] [Google Scholar]
  • 41. Delaunay F, Khan A, Cintra A, et al. Pancreatic β cells are important targets for the diabetogenic effects of glucocorticoids. J Clin Invest. 1997;100:2094–2098 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Ranta F, Avram D, Berchtold S, et al. Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4. Diabetes. 2006;55:1380–1390 [DOI] [PubMed] [Google Scholar]
  • 43. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343:230–238 [DOI] [PubMed] [Google Scholar]
  • 44. Frosi Y, Anastasi S, Ballaro C, et al. A two-tiered mechanism of EGFR inhibition by RALT/MIG6 via kinase suppression and receptor degradation. J Cell Biol. 2010;189:557–571 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Ying H, Zheng H, Scott K, et al. Mig-6 controls EGFR trafficking and suppresses gliomagenesis. Proc Natl Acad Sci USA. 2010;107:6912–6917 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Scheving LA, Buchanan R, Krause MA, Zhang X, Stevenson MC, Russell WE. Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes. Am J Physiol Gastrointest Liver Physiol. 2007;293:G552–559 [DOI] [PubMed] [Google Scholar]
  • 47. Blandino-Rosano M, Perez-Arana G, Mellado-Gil JM, Segundo C, Aguilar-Diosdado M. Anti-proliferative effect of pro-inflammatory cytokines in cultured β cells is associated with extracellular signal-regulated kinase 1/2 pathway inhibition: protective role of glucagon-like peptide -1. J Mol Endocrinol. 2008;41:35–44 [DOI] [PubMed] [Google Scholar]
  • 48. Rescan C, Coutant A, Talarmin H, et al. Mechanism in the sequential control of cell morphology and S phase entry by epidermal growth factor involves distinct MEK/ERK activations. Mol Biol Cell. 2001;12:725–738 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Talarmin H, Rescan C, Cariou S, et al. The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G1 phase progression in proliferating hepatocytes. Mol Cell Biol. 1999;19:6003–6011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Jin N, Gilbert JL, Broaddus RR, Demayo FJ, Jeong JW. Generation of a Mig-6 conditional null allele. Genesis. 2007;45:716–721 [DOI] [PubMed] [Google Scholar]

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