SAT064 Establishment And Initial Characterization Of Two Parallel Targeted Human ZnT8 Knock-in Murine Lines For Studying The Type 2 Diabetes Risk Variant At SNP rs13266634 Of The ZnT8 Gene
Authors: Wang Janice C , Adelma Cade , Hayes M Geoffrey , El Muayed Malek 
Published: October 2023

Disclosure: J.C. Wang: None. C. Adelma: None. M.G. Hayes: None. M. El Muayed: None.

Background: Studies showed an association between R variant at position 325 in the β-cell zinc transporter ZnT8 and an increased risk for developing type 2 diabetes mellitus (T2DM). However, the mechanism by which this variant confers an increased risk for type 2 diabetes remains unclear. Methods: We generated a murine model for studying the effect of SNP rs13266634 on β-cell physiology. This model consists of two parallel murine lines in which conditional (RIP-Cre induced) expression constructs for the human variants of SLC30A8 (ZnT8), namely R (hZnT8R325, T2DM risk allele) and W (hZnT8W325, non-risk allele) of SNPrs13266634 were inserted into the rosa26 locus via targeted integration. β-cell specific expression of each of ZnT8R325 and ZnT8W325 was induced by cross breeding with a RIP-Cre line, expressing Cre recombinase in β-cells. All mouse lines were backcrossed into a C57BL/6N background null for the mouse variant of ZnT8. Islet zinc concentrations was measured by ICP-MS following hydrolysis in HNO3. Ex-vivo Glucose Stimulated Insulin Secretion Ratio (GSISR) was measured by incubation of isolated islets in 2.8 mM glucose, followed by 16.7 mM glucose minutes after an initial 60 min equilibration phase and calculating the insulin secretion at 16.7 mM/2.8 mM glucose. Data are reported as averages ± SEM from 3 independent experiments (n=3), each with 4 technical replicates. Results: Expression of both variants was verified by mRNA PCR from islets. Levels of expression in both lines were identical, owing to the targeted integration approach. Expression of each of hZnT8R325 and ZnT8W325 resulted in restoration of islet zinc concentration to levels similar to those found in normal human islets and wild type mouse islets. This confirms proper function of the hZnT8R325 transgenes in this newly established model. hZnT8R expressing islets showed a trend to lower insulin response to ex-vivo glucose stimulation compared to hZnT8W325 expressing islets (GSISR- 1.37 ± 0.41 vs 1.97 ± 0.32 respectively), as well as Cre expressing (GSISR 1.53 ± 0.40) or inactive construct controls (GSISR 2.43 ± 0.57). In-vivo glucose stimulated insulin secretion was not significantly different between hZnT8R325 expressing mice and those expressing hZnT8W325. As previously reported by others, all mice with β-cell Cre expression (RIP-Cre activated hZnT8 expressing lines and RIP-Cre controls) showed a lower insulin secretory response to glucose compared to non-Cre expressing mice (inactive construct controls). Conclusion: The newly created murine mouse model expresses functional human homologues of the ZnT8 variants hZnT8R325 and hZnT8W325 that result in restored beta cell zinc uptake. A trend to lower insulin secretory capacity was observed in ex-vivo glucose stimulated insulin secretion experiments. This model will be valuable tool to elucidate the functional consequences of the T2DM risk variant at SNP rs13266634.

Presentation: Saturday, June 17, 2023