Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic β cells recapitulates neonatal diabetes
Christophe A. Girard, … , Jens C. Brüning, Frances M. Ashcroft
Christophe A. Girard, … , Jens C. Brüning, Frances M. Ashcroft
Published December 8, 2008
Citation Information: J Clin Invest. 2009;119(1):80-90. https://doi.org/10.1172/JCI35772.
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Research Article Metabolism

Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic β cells recapitulates neonatal diabetes

Abstract

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic β cells. These β-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from β-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic β cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in β-V59M β cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic β cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. β-V59M islets also displayed a reduced percentage of β cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of β-V59M mice. Their cause requires further investigation.

Authors

Christophe A. Girard, F. Thomas Wunderlich, Kenju Shimomura, Stephan Collins, Stephan Kaizik, Peter Proks, Fernando Abdulkader, Anne Clark, Vicky Ball, Lejla Zubcevic, Liz Bentley, Rebecca Clark, Chris Church, Alison Hugill, Juris Galvanovskis, Roger Cox, Patrik Rorsman, Jens C. Brüning, Frances M. Ashcroft

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Figure 1

Generation and identification of transgenic mice.

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Generation and identification of transgenic mice. (A and B) Targeting st...
(A and B) Targeting strategy. Insertion of the targeting vector ROSA26-STOP-Kir6.2V59M (B) into the ROSA26 locus (A) by homologous recombination in mouse ES cells. (C) Targeted ROSA locus. (D) After Cre-mediated deletion of the STOP cassette, Kir6.2-V59M is expressed under the control of the endogenous ROSA26 promoter. 1–3, ROSA26 exons 1–3; SA, splice acceptor signal; PGK-Neo, neomycin resistance gene; STOP, transcriptional STOP signal; DT-A, diphtheria toxin A; pA, polyadenylation signal; E, EcoRI restriction site; P, PacI restriction site; FRT, flippase recombinase target site. (E) Southern blot analysis of targeted ES clones. Genomic DNA from G418-resistant or WT ES clones was digested with EcoRI and probed with a 5′ external ROSA probe (location indicated in A) to confirm targeting of the ROSA26 locus. In contrast with WT ES cells, the recombinant ES clone shows a 5.1-kb band expected from the targeted ROSA26 allele.