The MODY1 gene HNF-4α regulates selected genes involved in insulin secretion
Rana K. Gupta, Marko Z. Vatamaniuk, Catherine S. Lee, Reed C. Flaschen, James T. Fulmer, Franz M. Matschinsky, Stephen A. Duncan, Klaus H. Kaestner
Rana K. Gupta, Marko Z. Vatamaniuk, Catherine S. Lee, Reed C. Flaschen, James T. Fulmer, Franz M. Matschinsky, Stephen A. Duncan, Klaus H. Kaestner
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Article Metabolism

The MODY1 gene HNF-4α regulates selected genes involved in insulin secretion

Abstract

Mutations in the gene encoding hepatocyte nuclear factor-4α (HNF-4α) result in maturity-onset diabetes of the young (MODY). To determine the contribution of HNF-4α to the maintenance of glucose homeostasis by the β cell in vivo, we derived a conditional knockout of HNF-4α using the Cre-loxP system. Surprisingly, deletion of HNF-4α in β cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the mutant β cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60% reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays that the Kir6.2 gene is a transcriptional target of HNF-4α. Our data provide genetic evidence that HNF-4α is required in the pancreatic β cell for regulation of the pathway of insulin secretion dependent on the ATP-dependent potassium channel.

Authors

Rana K. Gupta, Marko Z. Vatamaniuk, Catherine S. Lee, Reed C. Flaschen, James T. Fulmer, Franz M. Matschinsky, Stephen A. Duncan, Klaus H. Kaestner

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Derivation of β cell–specific HNF-4α knockout mice. (A) HNF-4αloxP/loxP ...
Derivation of β cell–specific HNF-4α knockout mice. (A) HNF-4αloxP/loxP mice in which exon 2 was flanked by loxP sites were bred to Ins.Cre-transgenic mice expressing Cre recombinase under control of the rat insulin promoter. The resulting HNF-4αloxP/+; Ins.Cre offspring were then mated with HNF 4αloxP/loxP homozygotes to obtain HNF-4αloxP/loxP; Ins.Cre mutants and their littermate controls: HNF-4αloxP/+, HNF-4αloxP/loxP, and HNF-4αloxP/+; Ins.Cre. (B) Primers 1, 2, and 3 (red, blue, and green in A) were used for PCR genotyping of isolated islets from HNF-4αloxP/loxP; Ins.Cre and HNF-4αloxP/loxP mice. In the absence of Cre, amplification by primers 1 and 2 results in a 620-bp product. Cre-mediated excision of exon 2 results in a 450-bp product amplified by primers 1 and 3. Quantification of the bands shows that deletion occurs in approximately 70% of all islet cells (note that non–β cells make up 20–30% of the islet cell numbers). (C) Concordant with the results in B, mRNA levels of HNF-4α were reduced by 63% in mutant islets, as determined by quantitative PCR using primers specific to exon 2. *P < 0.05; n = 3 per group. (D and E) Immunostaining of pancreatic sections from adult control (D) and mutant (E) mice using an antibody against HNF-4α indicates that the number of β cells expressing HNF-4α protein is reduced by approximately 85–90% (arrow) in the mutant mouse. Non–β cells in the islet mantle still express HNF-4α protein (arrowhead) in the mutant mouse. Thus, HNF-4α is deleted efficiently and specifically in pancreatic β cells. Magnification, ×200.