WISHto analyse the effect of chronic β-casomorphin exposure on insulin domain of expression. (A) Insulin domain of exposure (black arrowheads) in WT zebrafish embryos exposed to 50 µg/ml of bBCM-5, bBCM-7, hBCM-5 and hBCM-7 from 3–6 dpf. (B) Statistical analysis of insulin domain of expression using Student's t-test. Fold change (compared to untreated control zebrafish embryos) shows insulin domain of expression significantly decreased following bBCM-5 or -7 exposure and significantly increased following hBCM-5 or -7 exposure. Data represent mean±s.e.m.; *P<0.05, **P<0.005; Student's t-test (n=20).

WISH to analyse the effect of chronic β-casomorphin exposure on glucagon and somatostatin domain of expression. (A) Glucagon (black arrowheads) and somatostatin (black arrows) domain of exposure in WT zebrafish embryos exposed to 50 µg/ml of bBCM-5, bBCM-7, hBCM-5 and hBCM-7 from 3–6 dpf. (B) Statistical analysis of glucagon and somatostatin domain of expression using Student's t-test. Fold change (compared to untreated control zebrafish embryos) shows glucagon domain of expression significantly increased following bBCM-7 exposure. No significant change in glucagon and somatostatin domain of exposure was detected following bBCM-5, hBCM-5 and -7 exposure. Data represent mean±s.e.m.; *P<0.05; Student's t-test (n=10).

WISH analysis of the opioid nature of the β-casomorphins. (A) WT zebrafish embryos were pre-treated with 10 μM naloxone for 2 h to block the μ-opioid receptors and exposed to naloxone and 50 µg/ml of bBCM-5, bBCM-7, hBCM-5 and hBCM-7 every 24 h from 3–6 dpf. (Black arrowheads represent the insulin domain of expression). (B) Statistical analysis of the insulin domain of expression using Student's t-test. Fold change (compared to untreated control zebrafish embryos) shows insulin domain of expression significantly decreased following bBCM-5, -7 exposure and significantly increased following hBCM-5, -7 exposure. WT embryos treated with µ-opioid receptor antagonist, naloxone and bBCM-5, -7 and hBCM-5, -7 showed no changes in the insulin domain of expression compared to untreated controls. Data represent mean±s.e.m.; *P<0.05, **P<0.005; Student's t-test. (n=20).

Effect of β-casomorphins on β-cell regeneration. (A) β-cell ablation in Tg (ins: CFP-NTR was performed using 12 mM MTZ from 3–4 dpf. No positively fluorescent cells were detected at 4 dpf indicating complete ablation (black arrowheads indicate β-cells). (B) Tg (ins: CFP-NTR) zebrafish embryos were treated with 12 mM MTZ from 3–4 dpf to ablate the pancreatic β and exposed to daily 24 h treatments of bBCM-5, bBCM-7, hBCM-5 and hBCM-7 from 4–7 dpf to analyse the effects of β-casomorphins on β-cell regeneration (black arrowheads indicate β-cells). (C) Statistical analysis using Student's t-test revealed a significant decrease in the area of CFP fluorescence (measured using Fiji) following treatment with bBCM-5 and bBCM-7 compared to ablated controls at 7 dpf. No changes were detected in the area of CFP fluorescence following treatment with hBCM-5 and hBCM-7 compared to ablated controls at 7 dpf. Data represent mean±s.e.m.; *P<0.0005; Student's t-test. (n=20).