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, 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
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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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 2

β-V59M mice express similar mRNA levels of WT and mutant Kir6.2.

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β-V59M mice express similar mRNA levels of WT and mutant Kir6.2. (A) Kir...
(A) Kir6.2 transcripts amplified by RT-PCR from islets isolated from 5-week-old β-V59M or WT mice were digested with BtsCI and loaded on a 2% agarose gel. Amplification of WT Kir6.2 cDNA generated a 282-bp product, which was cleaved by BtsCI into 177-bp and 105-bp products. Introduction of the V59M point mutation removed the unique BtsCI restriction site of the 282-bp amplicon, preventing its cleavage. Lanes 1 and 3, islets isolated from 2 different β-V59M mice. Lanes 2 and 4, islets isolated from 2 different WT mice. Lanes 5 and 6, positive controls using WT Kir6.2 and Kir6.2-V59M plasmids. In contrast with WT islets, β-V59M islets expressed the Kir6.2-V59M mutant gene, as demonstrated by the presence of a 282-bp BtsCI-resistant PCR product. (B) Lanes 1 and 2, RT-PCR of GFP using cDNA prepared from WT or β-V59M islets (isolated from β-V59M [1] and WT [1] mice shown in A, respectively). Lane 3, negative control. Lane 4, positive control using a plasmid-expressing GFP. GFP expression in β-V59M islets is demonstrated by amplification of a 328-bp product, which is absent in control islets. (C) Kir6.2 and SUR1 mRNA levels in islets isolated from 3-week-old WT or β-V59M mice, measured by quantitative PCR, and normalized to β2-microglobulin mRNA levels. Results are expressed as fold difference versus WT mice of the same litter. Results represent the mean ± SEM of 3 mice per genotype.