Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin

Abstract

One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The glucose-lowering effect of the treatment might play an important role in the regeneration process.

Fig 1. Scheme of the experimental design.

Fig 2. EGF/G treatment restores glycemia and stimulates beta cell regeneration after alloxan-induced ablation.

Non-fasting glycemia (A) and body weight (B) were monitored; n = 24–37 per group. (C) Beta cell mass, (D) number of insulin-positive cells per islet, (E) islet density (number of insulin-positive clusters per mm²) and (F) beta cell size were assessed in CTRL, ALX and ALX+EGF/G on d3 and d8 post-alloxan. For histological analysis n = 4–7 per group per time point. (G) Fasted and fed plasma C-peptide levels (day 3) were significantly reduced in ALX+EGF/G compared to CTRL. (H) Fasting glycemia on day 3; n = 3 per group per time point. Symbol * represents the statistical significance of each condition compared to CTRL. The horizontal bar denotes the significant difference between the experimental groups. *,+, P < 0.05; **,++, P < 0.01; ***,+++, P < 0.001.

Fig 3. In vitro effect of EGF and/or gastrin on glucose uptake in C2C12 myocytes.

The percentage glucose uptake activity when treated with EGF alone, gastrin alone (A) or EGF+gastrin (B). Symbol * represents the statistical significance of each condition compared to positive control (1μM Ins); **, P < 0.01; ***, P < 0.001.

Fig 4. Beta cell lineage tracing: Contribution of beta cell neogenesis.

(A) The percentage of X-gal-labeled cells that co-express insulin. Beta cell tracing in RIP-CreER/R26-LacZ mice showed that almost all X-gal-positive cells are insulin positive in CTRL. A decrease of insulin positivity was observed in ALX and ALX+EGF/G on d3. Insulin positivity was recovered in ALX+EGF/G on d8. (B) X-gal-labeling of total beta cells (percentage beta cells labeled with X-gal). Total beta cell population includes INS^- beta cell fraction. Percentage labeled beta cells in ALX and ALX+EGF/G was significantly reduced compared to CTRL on d8. (C) Pancreata of RIP-CreER/R26-LacZ mice were stained for insulin and X-gal. (D) The percentage insulin-positive clusters labeled with X-gal. The percentage X-gal-labeled insulin-positive clusters remained unchanged between d3 and d8. n = 4–6 per group per time point. Symbol * represents the statistical significance of each condition compared to CTRL. The horizontal bar denotes the significant difference between the experimental groups. +, P < 0.05; **, P < 0.01; ***,+++, P < 0.001.

Fig 5. Acinar cell lineage tracing: No important contribution of acinar cells.

(A) The percentage AMY⁺acinar cells labeled with YFP. Acinar cell tracing in Ela-CreERT/R26-YFP mice showed comparable percentages YFP-labeled AMY⁺acinar cells in CTRL, ALX and ALX+EGF/G. (B) The percentage of INS⁺beta cells labeled with YFP. Labeling index of beta cells show no contribution of acinar cells in newly generated beta cells. (C) Pancreata of Ela-CreERT/R26-YFP mice were stained for insulin and YFP. n = 3–4 per group. No statistical difference (P ≥ 0.05) was found between the conditions.

Fig 6. No evidence for an important contribution of alpha cells.

(A) IHC for insulin and glucagon. (B) The percentage of glucagon-positive cells that co-express insulin. Transitional cells co-expressing glucagon and insulin were not observed. There were no significant differences in (C) alpha cell mass and (D) number of alpha cells per islet in ALX and ALX+EGF/G on d3 and d8 compared to CTRL; n = 4–5 per group per time point. No statistical difference (P ≥ 0.05) was found between the conditions.

Fig 7. Beta cell proliferation and macrophage infiltration.

(A) Continuous BrdU-labeling from day 3 to day 8 showed a significant increase in proliferating beta cells in ALX and ALX+EGF/G compared to CTRL. (B) Alloxan-induced beta cell ablation promotes F4/80⁺macrophage infiltration (number of F4/80⁺cells per mm²) in ALX and ALX+EGF/G on d3. EGF/G suppresses further infiltration on d8. (C) Pancreata stained for insulin and F4/80 showed reduced peri- and intra-islet infiltration in ALX+EGF/G on d8. n = 3 per group per time point. Symbol * represents the statistical significance of each condition compared to CTRL. The horizontal bar denotes the significant difference between the experimental groups. *,+, P < 0.05; **,++, P < 0.01; ***, P < 0.001.

Fig 8. Estimation of relative contribution to the beta cell mass of pre-existing and neogenic beta cells.