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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Figure 6

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HNF-4α directly activates the Kir6.2 gene. (A) The consensus binding sit...
HNF-4α directly activates the Kir6.2 gene. (A) The consensus binding site for HNF-4α is 13 bp long and was derived from 71 known HNF-4α binding sequences from the literature (55) using the program Weblogo (http://weblogo.berkeley.edu/). The size of the letters reflects the frequency at which the nucleotide appears at that position in the binding site. (B) Putative HNF-4α binding site in the Kir6.2 promoter identified using NUBIScan, which uses a transcription factor–binding site–identification algorithm to identify nuclear receptor binding sites. Note that the site located at position –2,300 matches all determinant nucleotides in the HNF-4α consensus site shown in A. (C) EMSA demonstrates that HNF-4α binds to the identified binding site in the Kir6.2 gene as well as the HNF-4α consensus site. In supershift experiments using 2 different antibodies raised against HNF-4α, the identity of the bound protein is confirmed to be HNF-4α. (D) Cotransfection of BHK cells with HNF-4α and pGL3-Kir6.2, expressing luciferase under the control of the 237-bp region of Kir6.2 containing the binding site, results in a dose-dependent increase in luciferase activity, indicating that this element serves as an HNF-4α–dependent enhancer. Mutation of this binding site abolishes the transcriptional activation. Statistical analysis was performed by ANOVA; n = 3 for each transfection condition.