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Overby, Peter

University of British Columbia

Pancreatic β-cells β-cells are critical for systemic glucose homeostasis, and most of them undergo cell death during the pathogenesis of type 1 diabetes. We previously showed that a Na⁺ channel inhibitor, carbamazepine, could protect β-cells in vitro and in vivo. To define the molecular mechanism of carbamazepine actions in the context of type 1 diabetes, we developed new mouse strains including non-obese diabetic (NOD):Ins1ᶜʳᵉ mice. Comparison of wildtype (Ins1ʷᵗᐟʷᵗ), Ins1 heterozygous (Ins1ᶜʳᵉᐟʷᵗ or Ins1ᴺᵉᵒᐟʷᵗ), and Ins1 null (Ins1ᶜʳᵉ/ᴺᵉᵒ) littermates on a NOD background, showed that female Ins1ᴺᵉᵒᐟʷᵗ mice exhibited significant protection from diabetes, with further near-complete protection in Ins1ᶜʳᵉᐟʷᵗ mice. To investigate the specific role of the Na⁺ channel gene, Scn9a, in β-cell function and survival, we generated a β-cell specific knockout of Scn9a on the NOD background by crossing the floxed Scn9a allele onto the Ins1ᶜʳᵉ knock-in mouse line, resulting in following genotypes: knockout (NOD.Ins1ᶜʳᵉ;Scn9aᶠˡᵒˣᐟᶠˡᵒˣ), heterozygous (NOD.Ins1ᶜʳᵉ;Scn9aᶠˡᵒˣᐟʷᵗ), and wildtype littermate controls (NOD.Ins1ᶜʳᵉ;Scn9aʷᵗᐟʷᵗ). Indeed, we observed near complete ablation of Na⁺ currents in knockout β-cells, and intermediate Na⁺ currents in the heterozygotes. Glucose-stimulated insulin release was significantly reduced from NOD.Ins1Cre;Scn9aʷᵗᐟᶠˡᵒˣ and NOD.Ins1ᶜʳᵉ;Scn9aᶠˡᵒˣᐟᶠˡᵒˣ islets from both male and female mice. Carbamazepine also inhibited glucose-stimulated insulin secretion, from both mouse and human islets, and the effects of carbamazepine on insulin secretion in vitro were not additive to the effects of Scn9a knockout, suggesting that Scn9a is the target of carbamazepine in β-cells. Complete Scn9a deletion also protected β-cells from death in vitro, and non-additively to carbamazepine treatment. Because we could not assess type 1 diabetes incidence in NOD.Ins1ᶜʳᵉ;Scn9aᶠˡᵒˣᐟᶠˡᵒˣ without being confounded by the protective effect of the Cre recombinase knock-in and replacement of one Ins1 allele, we studied NOD.Scn9aᶠˡᵒˣᐟᶠˡᵒˣ after AAV8-Ins1ᶜʳᵉ virus delivery and their virus-injected NOD.Scn9aʷᵗᐟʷᵗ littermate controls. A significant protective effect of Scn9a deletion on diabetes incidence was measured. Our data show Scn9a plays important roles in β-cell function, but also contributes to β-cell death in the context of diabetes-relevant stresses and type 1 diabetes progression. Scn9a may therefore be a novel drug target to preserve β-cells in type 1 diabetes.

Text

2023-08-23

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85579

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/