Role of upstream stimulatory factor 2 in glutamate dehydrogenase gene transcription

Abstract

Glutamate dehydrogenase (Gdh) plays a central role in ammonia detoxification by catalysing reversible oxidative deamination of l-glutamate into α-ketoglutarate using NAD or NADP as cofactor. To gain insight into transcriptional regulation of , the gene that codes for Gdh, we isolated and characterised the 5' flanking region of from gilthead sea bream (). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver of were also addressed. 5'-Deletion analysis of promoter in transiently transfected HepG2 cells, electrophoretic mobility shift assays, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis allowed us to identify upstream stimulatory factor 2 (Usf2) as a novel factor involved in the transcriptional regulation of Analysis of tissue distribution of Gdh and Usf2 mRNA levels by reverse transcriptase-coupled quantitative real-time PCR (RT-qPCR) showed that Gdh is mainly expressed in the liver of, while Usf2 displayed ubiquitous distribution. RT-qPCR and ChIP assays revealed that long-term starvation down-regulated the hepatic expression of Gdh and Usf2 to similar levels and reduced Usf2 binding to promoter, while refeeding resulted in a slow but gradual restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivates by binding to an E-box located in the proximal region of promoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation of transcription in the fish liver.

Uncoupling Thermotolerance and Growth Performance in Chinook Salmon: Blood Biochemistry and Immune Capacity

“…Glutamate dehydrogenase (GDH) reversibly catalases the oxidative deamination of glutamate to produce α-ketoglutarate (as an energy substrate for the Krebbs cycle) and ammonia, playing a key role in nitrogen excretion in fish [ 51 ]. The lower GDH activity ( Figure 3 A: 16) (and mean urea levels ( Figure 3 B: 12)) in the weight-loss groups indicates limited protein catabolism.…”

Multiomics analyses identified epigenetic modulation of the S100A gene family in Kawasaki disease and their significant involvement in neutrophil transendothelial migration

“…However, promoter is a rough and ambiguous region relative to the TSS of a gene. Although long genomic fragments were defined as putative promoter regions in studies [ 21 , 22 ], the functional domains confirmed by experiment or selected for experiment were usually close to the TSSs [ 34 – 36 ]. Such phenomenon was also consistent with our finding in Fig.…”

Metformin counteracts glucose-dependent lipogenesis and impairs transdeamination in the liver of gilthead sea bream (Sparus aurata)

“…Indeed, optimal growth of fish requires high levels of dietary protein, and amino acids are the most potent insulin secretagogues in fish (34). Therefore, transdeamination, which involves transferring of amino groups from amino acids to ␣-ketoglutarate to produce glutamate and subsequent glutamate deamination by GDH, is very important in the fish liver for entering the carbon skeleton of amino acids into the TCA cycle to obtain energy and for biosynthetic purposes (7,14,20,22,38). The fact that metformin markedly decreased GDH expression, and to a lesser extent ALT activity, suggests reduced amino acid deamination and transamination capacity in the liver of S. aurata, and, therefore, limited use of amino acids for gluconeogenic purposes, which, in turn, may be essential for the long-term hypoglycemic effect of metformin in the liver of S. aurata.…”