Ablation of pancreatic β-cells induced depletion of insulin production in ins:epNTR-mCherry zebrafish. (A) Schematics of the transgenic zebrafish showing the ablation of pancreatic β-cells using epNTR-mediated chemogenetic ablation system. (B,C) In-vivo fluorescence images showing pancreatic β-cells in Ins:epNTR-mCherry larvae treated with DMSO (B) and MTZ (C) at 9 dpf. (D) Quantification of fluorescence intensity in mcherry-expressing pancreatic β-cells from panel (B) and (C), ****p < 0.0001, t-test (n = 12 larvae per group). (E) Quantification of expression of ins mRNA in transgenic larvae, ****p < 0.0001, t-test (n = 15 larvae per group). Scale bar, 20 μm in (B).

Depletion of insulin production caused peripheral neuropathy in the peripheral lateral line nerve of ins:epNTR-mCherry zebrafish. (A–C)In-vivo fluorescence images showing sensory nerve endings in Ins:epNTR-mCherry expressing Tg (tubb2b:dsred) larvae treated with DMSO (A) and MTZ (B) at 9 dpf. In-vivo fluorescence images showing sensory nerve endings in Tg (tubb2b:dsred) siblings treated with MTZ (C) at 9 dpf. (D) Quantification of the number of axon bifurcation from panel (A–C), ****p < 0.0001, one-way ANOVA (n = 12 larvae per group). (E–F’) Fluorescence images showing sensory nerve endings with postsynaptic MAGUK⁺ puncta in Ins:epNTR-mCherry expressing Tg (tubb2b:dsred) larvae treated with DMSO (E,E’) and MTZ (F,F’) at 9 dpf. (G) Quantification of fluorescence intensity of MAGUK⁺ puncta in the transgenic larvae presented in panels (E’) and (F’), ****p < 0.0001, t-test (n = 12 in DMSO group and n = 10 in MTZ group). Scale bar, 10 μm in (A) and (E).

Depletion of insulin production results in defective rheotaxis behavior, but not locomotor behavior, in ins:epNTR-mCherry zebrafish. (A) Quantification of the distribution of the Ins:epNTR-mCherry larvae performing rheotactic angles following treatment with DMSO and MTZ, and siblings treated with MTZ at 9 dpf, ***p = 0.0004, Chi-squared test (n = 15 larvae per group). (B) Quantification of the percentage of the transgenic larvae showing rheotactic behavior following treatment with DMSO and MTZ, and siblings with MTZ at 9 dpf, **p = 0.0064, Chi-squared test (n = 15 larvae per group). (C) Tracking visualization of the transgenic larvae performing free locomotion in light state at 9 dpf. (D,E) Quantification of the distance moved (mm) (D) and velocity (mm/s) (E) from the transgenic larvae performing free locomotion under light conditions following treatment with DMSO and MTZ, and sibling with MTZ at 9 dpf (n = 32 larvae per group).

Depletion of insulin production elevates glucose levels, but not ROS, in ins:epNTR-mCherry zebrafish. (A) Quantification of the glucose levels (mg/dL) in Ins:epNTR-mCherry larvae treated with DMSO and MTZ at 9 dpf, ****p < 0.0001, t-test (n = 20 larvae per one data point, total n = 100 larvae per group). (B) Quantification of the ROS intensity in the PLLg cells of Ins:epNTR-mCherry expressing Tg (tubb2b:dsred) larvae presented in panels (D’) and (E’) (n = 10 larvae per group). (C) Quantification of the number of PLLg cells of the transgenic larvae presented in panels (D”) and (E”) (n = 11 larvae per group). (D–E”)In-vivo fluorescence images showing PLLg cell and CellROX ROS detector in the transgenic larvae treated with DMSO (D–D”) or MTZ (E–E”) at 9 dpf. Scale bar, 20 μm in (D).

BMS-754807-induced IR inhibition reduces PLL nerve endings in ins:epNTR-mCherry zebrafish. (A–C). In-vivo fluorescence images showing sensory nerve endings in Ins:epNTR-mCherry expressing Tg (tubb2b:dsred) larvae treated with DMSO (A), MTZ, (B) and BMS-754807 (C) at 9 dpf. (D) Quantification of the number of axon bifurcation as depicted in panels (A–C), ****p < 0.0001, one-way ANOVA (n = 12 larvae in the DMSO group, n = 13 larvae in the MTZ group, and n = 16 larvae in the inhibitor group). Scale bar, 10 μm in (A).

Depletion of insulin production downregulates the mTOR signaling pathway in the PLLg in ins:epNTR-mCherry zebrafish. (A–B”) Fluorescence images showing the PLLg and pS6⁺ cells in Ins:epNTR-mCherry expressing Tg (tubb2b:dsred) larvae treated with DMSO (A–A”) and MTZ (B–B”) at 9 dpf. (C) Quantification of the number of pS6⁺ PLLg cells from panels (A) and (B), *p = 0.0305, t-test (n = 10 larvae per group). Scale bar, 20 μm in (A).