Oligonucleotide Microarray Analysis Reveals PDX1 as an Essential Regulator of Mitochondrial Metabolism in Rat Islets

Benoit R Gauthier,Thierry Brun,Eve Julie Sarret,Hisamitsu Ishihara,Olivier Schaad,Patrick Descombes,Claes B Wollheim

doi:10.1074/jbc.m405030200

Benoit R Gauthier, Thierry Brun + Show 5 more

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https://doi.org/10.1074/jbc.m405030200

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Abstract

Mutations in the transcription factor IPF1/PDX1 have been associated with type 2 diabetes. To elucidate beta-cell dysfunction, PDX1 was suppressed by transduction of rat islets with an adenoviral construct encoding a dominant negative form of PDX1. After 2 days, there was a marked inhibition of insulin secretion in response to glucose, leucine, and arginine. Increasing cAMP levels with forskolin and isobutylmethylxanthine restored glucose-stimulated insulin secretion, indicating normal capacity for exocytosis. To identify molecular targets implicated in the altered metabolism secretion coupling, DNA microarray analysis was performed on PDX1-deficient and control islets. Of the 2640 detected transcripts, 70 were up-regulated and 56 were down-regulated. Transcripts were subdivided into 12 clusters; the most prevalent were associated with metabolism. Quantitative reverse transcriptase-PCR confirmed increases in succinate dehydrogenase and ATP synthase mRNAs as well as pyruvate carboxylase and the transcript for the malate shuttle. In parallel there was a 50% reduction in mRNA levels for the mitochondrially encoded nd1 gene, a subunit of the NADH dehydrogenase comprising complex I of the mitochondrial respiratory chain. As a consequence, total cellular ATP concentration was drastically decreased by 75%, and glucose failed to augment cytosolic ATP, explaining the blunted glucose-stimulated insulin secretion. Rotenone, an inhibitor of complex I, mimicked this effect. Surprisingly, TFAM, a nuclear-encoded transcription factor important for sustaining expression of mitochondrial genes, was down-regulated in islets expressing DN79PDX1. In conclusion, loss of PDX1 function alters expression of mitochondrially encoded genes through regulation of TFAM leading to impaired insulin secretion.

Highlights

Type 2 diabetes mellitus is a common severe disease of intermediary metabolism usually caused by both ␤-cell dysfunction and resistance to the biological actions of insulin on its main target tissues
To ensure functionality of the adenoviral construct, the insulinoma cell line INS-1E (30,000 cells) was infected with 4 and 17 ϫ 106 viral particles of either AdCaLacZ or AdRIPDN79PDX1, and mRNA levels for insulin as well a GLUT2 were quantified by real time RT-PCR
These results suggest that DN79PDX1 is expressed and capable of interacting with a cognate DNA-binding site in islets infected with the adenoviral construct AdRIPDN79PDX1

Summary

Introduction

Type 2 diabetes mellitus is a common severe disease of intermediary metabolism usually caused by both ␤-cell dysfunction and resistance to the biological actions of insulin on its main target tissues (liver, muscle, and fat). Inactivation of both pdx alleles in murine models results in pancreas agenesis, whereas heterozygous mice or animals carrying a ␤-cell-specific deletion of the gene exhibit impaired glucose tolerance (8 –11). Several of these models develop overt diabetes with age indicating a progressive deterioration of ␤-cell function and/or mass. DNA microarray technology has become a powerful tool to decipher the complex genetic networks altered in response to environmental insults and disease We use this technology to study the impact of PDX1 on ␤-cell physiology by comparing global gene profiles of islets infected with a viral vector expressing a dominant negative variant of PDX1 (AdRIPDN79PDX1) to control LacZ-infected islets. Our study provides a novel role of PDX1 in mitochondrial metabolism through regulation of mitochondrial genes

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