The majority of neonatal diabetes (NDM), defined by early onset of hyperglycemia due to impaired β-cell function, is caused by gain-of-function (GOF) mutations in the ATP-sensitive potassium (KATP) channel genes (ABCC8 encoding SUR1 and KCNJ11 encoding Kir6.2). These GOF mutations lead to increased KATP channel activity, decreased β-cell excitability, and impaired insulin secretion. A zebrafish model of NDM can provide a high throughput platform for understanding mechanisms of NDM and testing potential therapeutic approaches. To create such a model, we used CRISPR mutagenesis to introduce the most common human Kir6.2 GOF mutation (R201H) into the equivalent zebrafish locus. Patch-clamp analysis demonstrates decreased ATP sensitivity in β-cells isolated from mutant fish (K1/2 for ATP inhibition = 17.21 ±1.66 mM in WT, 40.38 ± 7.6 mM in heterozygous R201H). Increasing [glucose] causes a robust increase of [Ca]i in WT islets expressing a GCaMP6s fluorescent reporter, but there is no glucose-dependent increase in R201H islets. R201H fish have smaller overall body size, indicative of failure to thrive, and β-cell density is reduced ∼50% in R201H compared to WT. Whole body glucose was measured from larval zebrafish using a fluorometric assay. Starting at 4 weeks of age, blood glucose was directly measured by glucometer from the transected cardiac cavity. While R201H animals are markedly hyperglycemic (BG > 250 mg/dl) at 4 weeks, blood sugar normalizes (BG <120 mg/dl) by ∼12 weeks of age.

In conclusion, R201H mutants demonstrate the expected KATP GOF, loss of glucose-dependence of [Ca]i, and marked hyperglycemia early in life, proving a viable model of the molecular and cellular consequences of NDM. Intriguingly, early hyperglycemia is followed by normalization of blood sugar in older fish. This resolution of hyperglycemia is an unexpected finding, and may provide further insight into physiologic differences between transient and permanent NDM.


J. Ikle: None. S.S. Singareddy: None. R.C. Tryon: None. N. York: None. L. Yin: None. W. Chen: None. C. Nichols: None.


National Institutes of Health (DK109407); Pediatric Endocrine Society; Endocrine Fellows Foundation

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