Genome-edited zebrafish model of ABCC8 loss-of-function disease

Abstract

ATP-sensitive potassium channel (K_ATP)gain- (GOF) and loss-of-function (LOF) mutations underlie human neonatal diabetes mellitus (NDM) and hyperinsulinism (HI), respectively. While transgenic mice expressing incomplete K_ATP LOF do reiterate mild hyperinsulinism, K_ATP knockout animals do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis are regulated by identical K_ATP channels in zebrafish. SUR1 truncation mutation (K499X) was introduced into the abcc8 gene to explore the possibility of using zebrafish for modeling human HI. Patch-clamp analysis confirmed the complete absence of channel activity in β-cells from K499X (SUR1^-/-) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish nor in homozygous SUR1^-/- fish, mimicking findings in SUR1 knockout mice. Mutant fish did, however, demonstrate impaired glucose tolerance, similar to partial LOF mouse models. In paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent LOF mutations, these gene-edited animals provide valid zebrafish models of K_ATP -dependent pancreatic diseases.

Keywords: KATP; calcium channels; insulin secretion; metabolism; pancreas; zebrafish.

Graphical abstract

Figure 1.

Genome-modified zebrafish model of SUR1 LOF (a) Schematic of SUR1 protein structure showing functional domains and indicating location of premature stop mutation (K499X) within TMD1. (b) Body weight in WT (n = 11), heterozygous SUR1 mutants (n = 12) and SUR1 knockout (n = 12) fish at age 8 weeks. Density of β-cell within individual islets is not different between WT (n = 5) and SUR1 mutant (n = 15). (c) Fasting blood glucose over time shows no significant difference between homozygous mutants and controls (n = 11–16 per timepoint). (d) Blood glucose versus time in response to glucose load in SUR1^−/−, WT and SUR1+/- fish (n = 8–16 per timepoint).

Figure 2.

SUR1 LOF [499X] abolishes K_ATP channel activity (a) Representative K_ATP channel activity in inside-out patch clamp recordings from inside-out patch clamp recordings of WT (above) and homozygous SUR1[499X] (SUR1^−/−) mutant β-cells (below) in the presence of ATP (μM), or addition of diazoxide (mM), as indicated. Voltage was clamped at −50 mV. (b) KATP channel density in WT and SUR1-/- patches (n = 17,10).

Figure 3.

SUR1^−/− islets exhibit elevated basal [Ca2+] and reduced responsivity to glucose (a) Representative recordings of intracellular calcium in the presence of 2 mM glucose (2 G) and following switch to 20 mM glucose (20 G) and then 30 mM KCl (30 K), for WT (above, n = 6) and SUR1^−/− (below, n = 12). Fluorescence is normalized to maximum fluorescence in 30 K (f = 1), and minimum fluorescence (f = 0) anywhere within the record. Values for each islet are also shown. (b) Average calcium in each condition for WT (N = 8) and SUR1^−/− (n = 14). Data in B are analyzed by 1-way ANOVA followed by multiple unpaired t-tests. (*) p < .05, (**) p < .01.