Targeting polyamine biosynthesis to stimulate beta cell regeneration in zebrafish

Abstract

Type 1 diabetes (T1D) is a disease characterized by destruction of the insulin-producing beta cells. Currently, there remains a critical gap in our understanding of how to reverse or prevent beta cell loss in individuals with T1D. Previous studies in mice discovered that pharmacologically inhibiting polyamine biosynthesis using difluoromethylornithine (DFMO) resulted in preserved beta cell function and mass. Similarly, treatment of non-obese diabetic mice with the tyrosine kinase inhibitor Imatinib mesylate reversed diabetes. The promising findings from these animal studies resulted in the initiation of two separate clinical trials that would repurpose either DFMO (NCT02384889) or Imatinib (NCT01781975) and determine effects on diabetes outcomes; however, whether these drugs directly stimulated beta cell growth remained unknown. To address this, we used the zebrafish model system to determine pharmacological impact on beta cell regeneration. After induction of beta cell death, zebrafish embryos were treated with either DFMO or Imatinib. Neither drug altered whole-body growth or exocrine pancreas length. Embryos treated with Imatinib showed no effect on beta cell regeneration; however, excitingly, DFMO enhanced beta cell regeneration. These data suggest that pharmacological inhibition of polyamine biosynthesis may be a promising therapeutic option to stimulate beta cell regeneration in the setting of diabetes.

Keywords: Beta cell; DFMO; difluoromethylornithine; imatinib; islet; ornithine decarboxylase; polyamine biosynthesis; regeneration; type 1 diabetes; zebrafish.

Figure 1.

Whole-body development and exocrine pancreas size are unaltered with DFMO treatment. (A) A survival curve of zebrafish embryos treated with DFMO for 3 d (from 4 to 7 dpf). (B) Image of 7 dpf Tg(insa:flag-NTR:cryaa:mCherry);Tg(ptf1a:GFP) double transgenic control, NFP, and DFMO-treated zebrafish embryos. Scale bar = 1 mm. (C) Quantification of pancreas length normalized to whole-body length at 7 dpf. Each experiment was performed at least three times, starting with 15 embryos in each group. One-way ANOVA determined significance; data are represented as mean ± SEM * p < .05, ** p < .01, **** p < .0001. Tx, treatment.

Figure 2.

DFMO treatment enhances beta cell regeneration. (A) Representative immunofluorescence images of insulin- (green) and glucagon- (red) expressing cells in the principal islet of zebrafish embryos from each control and treatment group. Quantification of glucagon-expressing cells (B), insulin+/glucagon+ co-expressing cells (C), and insulin-expressing cells (D) in control and NFP-ablated zebrafish at 4 dpf as well as control, NFP and DFMO-treated zebrafish at 7 dpf. Each experiment was performed at least three times, starting with 15 embryos in each group. One-way ANOVA determined significance; data are represented as mean ± SEM * p < .05, ** p < .01, **** p < .0001. Tx, treatment; gluc, glucagon. Scale bars = 10 µm.

Figure 3.

Whole-body development and exocrine pancreas size are unaltered with Imatinib treatment. (A) A survival curve of zebrafish embryos treated with Imatinib for 3 d (from 4 to 7 dpf). (B) Quantification of pancreas length normalized to whole-body length at 7 dpf. Each experiment was performed at least 3 times, starting with 15 embryos in each group. One-way ANOVA determined significance. Data are represented as mean ± SEM ** p < .01, **** p < .0001. Tx, treatment.

Figure 4.

Imatinib treatment does not enhance beta cell regeneration. (A) Representative immunofluorescence images of insulin- (green) and glucagon- (red) expressing cells in the principal islet of zebrafish embryos treated with varying doses of Imatinib. (B) Representative immunofluorescence images of insulin- (green) and glucagon- (red) expressing cells in the principal islet of control and NFP-ablated zebrafish embryos at 4 and 7 dpf. Quantification of insulin-expressing cells (C), glucagon-expressing cells (D), and insulin+/glucagon+ co-expressing cells (E) in control and NFP-ablated zebrafish at 4 dpf as well as control, NFP and Imatinib-treated zebrafish at 7 dpf. Each experiment was performed at least 3 times, starting with 15 embryos in each group; one-way ANOVA determined significance; data are represented as mean ± SEM ** p < .01, **** p < .0001. Tx, treatment; gluc, glucagon. Scale bars = 10 µm.