Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression
Authors: Julieta M Ceruti
Eduardo T Cánepa
María E Scassa
Alejandra S Guberman
Journal: Journal of Biological Chemistry (2004)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: gene expression regulation; insulin; transcription factors; 5-aminolevulinate synthase (ALAS) gene; insulin-responsive region
Affiliations: Laboratorio de Biología Molecular, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II Piso 4, Ciudad Universitaria, 1428 Buenos Aires, Argentina.

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 HNF3beta or HNF3beta 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 HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta 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 HNF3beta 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. 

Retracted: No

PubPeer comments:
  TEM images of native soya protein nanoparticles and insulin loaded nanoparticles are shown in **Figure 3(a) and (b)**, respectively. Whereas the left image (a) shows aggregated nanoparticles of **unloaded soya protein**, the right one (b) depicts **insulin loaded scattered nanoparticles**. Both of the images reveal that the size of nanoparticles varies in the broad range from 40 to 200 nm. It is also clear from the TEM images that the unloaded nanoparticles have an irregular shape but while after loading of the insulin the soya protein nanoparticles acquire a little regular shape. It is also noteworthy that the state of aggregation is significantly reduced after insulin loading which may be attributed to the fact that the loading of insulin may cause weakening of inter-particle forces. ![file] Fig 3(a),(b) also feature in Fig. 5 of [Patel et al (2018)] as  Transmission electron micrograph images of **(a) native alginate powder, and (b) cypermethrin loaded Ca-alginate nanocarriers**. ![] ..while portions of 3(b) appear in Fig 3 A B of [Singh et al (2020)] as  TEM images of **CCIONPS and CYT loaded CCIONPs** . ![] ,