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

Abstract

Mutations in the transcription factor IPF1/PDX1 have been associated with type 2 diabetes. To elucidate ␤-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.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). The susceptibility for type 2 diabetes is inherited, but single diabetes genes have only been identified in about 5% of cases. Mutations in one of these genes, the homeodomain transcription factor ipf1 (also known as, pdx1, idx1, or stf1), have been associated with a rare form of maturity onset diabetes of the young (MODY4) 1 as well as predisposing individuals to late onset type 2 diabetes (1-5). Furthermore, homozygous null mutations in the ipf1 gene result in pancreas agenesis indicating that this transcription factor is indispensable for both pancreas development and subsequent ␤-cell function (6, 7).As in humans, inactivation of both pdx1 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 also develop overt diabetes with age indicating a progressive deterioration of ␤-cell function and/or mass. A recent study concluded that reduction in PDX1 expression impedes p...

The Fas pathway is involved in pancreatic β cell secretory function

“…The defective metabolism-secretion coupling may be a consequence of decreased PDX-1 expression. PDX-1 has recently been shown to be an essential regulator of several events in insulin secretion, including mitochondrial metabolism and in particular ATP production (18,20). In line with these findings, we observed a decrease in PDX-1 expression in the islets of Fas-deficient animals.…”

“…In particular, PDX-1 controls several events in glucose stimulated insulin secretion including mitochondrial metabolism (17)(18)(19)(20). Several observations point to a role for the Fas pathway in the regulation of cell cycle-independent events including T cell activation (21), renal tubular epithelial integrin function (22), and protection against neurodegeneration (23).…”

Targeting cyclophilin D and the mitochondrial permeability transition enhances β-cell survival and prevents diabetes in Pdx1 deficiency

“…Indeed, here and previously we observe caspase activation and increased TUNEL staining in Pdx1-deficient β-cells (9,16,26). Others have described transcriptional down-regulation of antiapoptotic factors and up-regulation of caspases by Pdx1 deficiency (27). However, activation of intrinsic pathway apoptosis by outer membrane permeabilization does not directly induce, nor require, dissipation of Δψ m as observed in Pdx1-deficient MIN6 cells.…”

Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis

“…In addition, viruses encoding truncated forms of XBP1 induced little (Ad-tXBP1s) or no apoptosis (Ad-XBP1u), and there was a close correlation between XBP1s activity and apoptosis, indicating that the increased apoptosis is specifically triggered by XBP1s. It is likely that the discrepancy between fibroblasts and beta cells is due to the role, discovered here, of XBP1s in inhibiting the expression of Pdx1 and Mafa, two key beta cell-specific transcription factors that are crucial for maintaining function [34] and, at least for Pdx1, survival [38,39]. Xbp1 knockdown partially reversed Pdx1 downregulation, but failed to prevent cytokine-or CPA-induced apoptosis, suggesting that Pdx1 downregulation is not a major determinant of beta cell apoptosis in response to cytokines or CPA.…”