Hepatic Nuclear Factor 3 and Nuclear Factor 1 Regulate 5-Aminolevulinate Synthase Gene Expression and Are Involved in Insulin Repression

Abstract

Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5 -regulatory region allowed us to identify an insulin-responsive region located at ؊459 to ؊354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3␤ or HNF3␤ plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3␤ is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3␤ can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3␤ binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Toward Heme: 5-Aminolevulinate Synthase and Initiation of Porphyrin Synthesis

“…125 An insulinresponsive region was identified at −459 bp to −354 bp, and this region was found to be required for insulin-mediated inhibition of the ALAS1 promoter. 126 The ALAS1 promoter also revealed motifs resembling consensus sequences for the binding of two nuclear proteins, hepatocyte nuclear factor 3β (HNF3β) and NF-1, involved in insulin repression of phosphoenolpyruvate carboxykinase (PEPCK) 127 and glucose transporter type 4 128 promoters, respectively. Insulin was not able to repress ALAS1 expression in HNF3-deficient HeLa cells, and HNF3β was shown to be involved in the basal expression of ALAS1.…”

“…Insulin was not able to repress ALAS1 expression in HNF3-deficient HeLa cells, and HNF3β was shown to be involved in the basal expression of ALAS1. 126 Furthering the implications of energy metabolism on ALAS1 expression, in 2005, Handschin et al 129 indicated that ALAS1 expression is regulated by the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α). PGC-1α was originally identified in brown fat tissue and was shown to be a cofactor that interacts with the nuclear receptor family member peroxisome proliferator-activated receptor γ (PPARγ).…”

Origins of interspecies variation in mammalian muscle metabolic enzymes

“…The following elements were identified in the PGC1␣ promoter based upon TRANSFACT and PATCH: specificity protein 1 element (GGCTGGG), cyclic AMPresponse element (GACGTCA), hepatocyte nuclear factor 3 element(TGTGTGC), inverted CCAAT element (ICE)/nuclear factor Y element (ATTGG), and E1 box (CATGTG). We also noted additional conserved sequences that have been identified (35) …”

Insulin Represses Transcription of the Thyroid Stimulating Hormone β-Subunit Gene through Increased Recruitment of Nuclear Factor I

“…Cells-Because NFI has been reported to mediate the repression of several genes by insulin (15,16), quantitative real-time PCR was employed to determine the affects of insulin on TSH␤ expression in thyrotroph cells via NFI. T␣T1 cells were cultured in the indicated concentrations of insulin, and the mRNA expression levels of the ␣GSU and TSH␤ were determined.…”

“…Insulin stimulates glucose uptake by many cells via a complex cascade of signaling events, and it affects GLUT4 gene transcription via NFI in adipose cells (13)(14)(15). Furthermore, NFI has been shown to mediate the repression of the 5-aminolevulinate synthase gene by insulin (16). However, no evidence has been presented regarding the direct influence of insulin on the binding of NFI protein to target gene promoters.…”