Differences in binding of hepatic nuclear proteins from lean and obese rats to the 5'-upstream region of tyrosine aminotransferase.
The glucocorticoid effects on liver tyrosine aminotransferase mRNA levels have been studied in young, lean, and obese Zucker (fa/fa) rats and 5'-upstream regions of the tyrosine aminotransferase (TAT) gene have been used in gel retardation studies to investigate nuclear protein binding. Hepatic TAT mRNA levels were increased in obese fa/fa rats but were normalized seven days after adrenalectomy. Corticosterone replacement to adrenalectomized rats restored the increased levels of TAT mRNA in the obese animals. A 60-bp fragment of upstream TAT DNA (-2463 to -2403) was identified which showed higher levels of band shifting after incubation with hepatic nuclear proteins of obese rats compared with the proteins from lean animals. This differential level of gel retardation was substantially reduced by alkaline phosphatase treatment of nuclear proteins. Gel retardation was reduced when nuclear proteins were prepared from adrenalectomized obese rats, and increased with nuclear proteins from adrenalectomized rats replaced with corticosterone. DNA affinity chromatography and gel electrophoresis identified three proteins of approximately 58, 62, and 65 kDa in the DNA-protein complex. Increased amounts of these three proteins were purified from nuclei of obese rats. HNF3 alpha antibodies induced hypershift of the gel retardation pattern implicating HNF3 alpha as one of the proteins that binds to the 60 bp DNA fragment. The data support the hypothesis that decreased phosphorylation of nuclear proteins in obese rats is glucocorticoid-dependent and may contribute to the altered transcriptional activity of glucocorticoid-responsive genes.
Dong
H
H
Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA.
Wang
Y
Y
Jenson
M
M
Braymer
H D
HD
York
D A
DA
D000315:Adrenalectomy; D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005938:Glucocorticoids / Q000502:physiology*; D008099:Liver / Q000201:enzymology*; D009687:Nuclear Proteins / Q000378:metabolism*; D009765:Obesity / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011924:Rats, Zucker; D014444:Tyrosine Transaminase / Q000235:genetics*
D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014444:Tyrosine Transaminase