Mig6 haploinsufficiency protects mice against streptozotocin-induced diabetes

Abstract

Aims/hypothesis: EGF and gastrin co-administration reverses type 1 diabetes in rodent models. However, the failure of this to translate into a clinical treatment suggests that EGF-mediated tissue repair is a complicated process and warrants further investigation. Thus, we aimed to determine whether EGF receptor (EGFR) feedback inhibition by mitogen-inducible gene 6 protein (MIG6) limits the effectiveness of EGF therapy and promotes type 1 diabetes development.

Methods: We treated Mig6 (also known as Errfi1) haploinsufficient mice (Mig6 (+/-)) and their wild-type littermates (Mig6 (+/+)) with multiple low doses of streptozotocin (STZ), and monitored diabetes development via glucose homeostasis tests and histological analyses. We also investigated MIG6-mediated cytokine-induced desensitisation of EGFR signalling and the DNA damage repair response in 832/13 INS-1 beta cells.

Results: Whereas STZ-treated Mig6 (+/+) mice became diabetic, STZ-treated Mig6 (+/-) mice remained glucose tolerant. In addition, STZ-treated Mig6 (+/-) mice exhibited preserved circulating insulin levels following a glucose challenge. As insulin sensitivity was similar between Mig6 (+/-) and Mig6 (+/+) mice, the preserved glucose tolerance in STZ-treated Mig6 (+/-) mice probably results from preserved beta cell function. This is supported by elevated Pdx1 and Irs2 mRNA levels in islets isolated from STZ-treated Mig6 (+/-) mice. Conversely, MIG6 overexpression in isolated islets compromises glucose-stimulated insulin secretion. Studies in 832/13 cells suggested that cytokine-induced MIG6 hinders EGFR activation and inhibits DNA damage repair. STZ-treated Mig6 (+/-) mice also have increased beta cell mass recovery.

Conclusions/interpretation: Reducing Mig6 expression promotes beta cell repair and abates the development of experimental diabetes, suggesting that MIG6 may be a novel therapeutic target for preserving beta cells.

Fig. 1

Proinflammatory cytokines induce MIG6 expression. (a) Human islets or (b) 832/13 cells were treated with cytokines, and MIG6/Mig6 expression was determined. n=4; *p<0.05. (c) INS2 mRNA levels from four human islet donors (± cytokines) were plotted against MIG6 mRNA levels (r²=0.40, p<0.05)

Fig. 2

MIG6 suppression rescues cytokine inhibition of EGFR. 832/13 cells were pretreated with cytokines for 16 h and then stimulated with EGF to determine phospho (p)-EGFR, EGFR and tubulin levels: (a) a representative image and (b) quantified results (white bars, no EGF; black bars, EGF treated). n=3, *p<0.05 vs untreated, †p<0.05 vs noncytokine EGF stimulated. The solid line indicates cropped lanes within the same experiment. (c–f) 832/13 cells were transduced with adenoviral vectors carrying either a scrambled control RNA (siCon) or shRNA sequence against Mig6 (siMig6). (c) MIG6 knockdown efficiency was determined. n=3, *p<0.05 vs siCon. Following transduction, cells were treated with cytokines for 8 h and then EGF, and p- EGFR, EGFR, p-ERK, ERK and tubulin levels were determined: (d) a representative image and (e–f) quantified results. n=3; *p<0.05 vs untreated, ^†p<0.05 vs siCon EGF stimulated

Fig. 3

Schematic representation of the experimental timeline

Fig. 4

Mig6^+/− mice are protected from STZ-induced glucose intolerance. GTT was performed 3 days post-STZ injection. (a) Time course and (b) incremental AUC are shown. Dotted lines indicate values for saline-injected Mig6^+/+controls. n=5; *p<0.05 vs STZ-Mig6^+/+

Fig. 5

STZ-Mig6^+/− mice have preserved beta cell function. (a, b) Circulating insulin levels during a glucose challenge (a) Time course and (b) AUC are shown. n=8, *p<0.05 vs STZ-Mig6^+/+. (c, d) Blood glucose levels during ITT pre- and post-STZ treatment. (e–g) Pdx1, IRS2, and Ins2 mRNA levels in islets isolated from saline-injected Mig6^+/+, STZ-Mig6^+/− and STZ-treated Mig6^+/+ mice. Dotted lines indicate values for saline-injected Mig6^+/+ controls. n>5; *p<0.05 vs STZ-Mig6^+/+

Fig. 6

MIG6 overexpression compromises beta cell integrity and islet function. (a–c) Isolated rat islets (n=3) were transduced with (a, b) CMV-GFP or CMV-MIG6 adenoviral vectors (*p<0.05 vs CMV-GFP) and (c) GSIS was assessed (white bars, low glucose; black bars, high glucose; *p<0.05 vs low glucose; ^†p<0.05 vs high glucose-stimulated CMV-GFP). (d–f) 832/13 cells were either transduced or not with CMV-GFP or CMV-MIG6 adenoviral vectors. Pdx1, IRS2 and Ins1/2 mRNA levels were determined. n=5, *p<0.05 vs untransduced or CMV-GFP

Fig. 7

MIG6 does not modulate the cytokine- or STZ-induced immune response. (a, b) Insulitis scoring (defined in Methods) of STZ-Mig6^+/− and STZ-Mig6^+/+ mouse pancreases. (a) Representative images of islets and (b) quantified results (white, 0; light grey, 1; dark grey, 2; black, 3). Scale bar, 100 µm. (c) Islets from Mig6^+/+ (white bars) and Mig6^+/− (black bars) were treated with cytokines and NO levels in islet culture medium was measured. n=3, *p<0.05 vs untreated

Fig. 8

STZ-Mig6^+/− mice show enhanced beta cell mass recovery. (a, b) Beta cell area was measured and expressed relative to pancreas area. Dotted line indicates the beta cell area of non-STZ-treated mice. n=8, *p<0.05 vs STZ-Mig6^+/+. (c, d) The percentage of pH3-positive (pH3⁺) beta cells in saline controls (white bars) and at 3 and 20 days post-STZ (grey and black bars, respectively). Arrows indicate proliferating beta cells. n>5, *p<0.05 vs non-STZ. (a, c) Representative images of beta cell mass at 20 days

Fig. 9

MIG6 promotes DNA damage-induced beta cell death. (a) 832/13 cells were treated with doxorubicin for 6 h or with cytokines for the indicated times. Phospho (p)-p53, p53 and tubulin protein levels were determined. (b–d) 832/13 cells were transduced with siCon or siMig6 adenoviral vectors, and treated with vehicle (white bars) or CA (black bars). p-p53, p53, caspase- 3 and GAPDH protein levels were determined. n=3, *p<0.05 vs untreated, ^†p<0.05 vs siCon+CA