RESUMO
Binding of sterol response element binding protein 1a to sterol response element-1 (SRE-1) in the promoter region of lanosterol 14 alpha-demethylase (14DM) has been demonstrated previously. Decreased 14DM activity has been shown to result in accumulation of the intermediate, 3 beta-hydroxy-lanost-8-en-32-al, a known translational downregulator of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Since it has also been demonstrated that feedback regulation of hepatic HMG-CoA reductase occurs primarily at the level of translation, the effects of dietary cholesterol and cholesterol lowering agents on levels of hepatic 14DM mRNA and immunoreactive protein were investigated. Addition of 1% cholesterol to a chow diet markedly decreased hepatic 14DM mRNA and protein levels in Sprague-Dawley rats. The extent and time course of this decrease in 14DM immunoreactive protein closely paralleled that of HMG-CoA reductase. Supplementation of the diet with the HMG-CoA reductase inhibitor, Lovastatin, to a level of 0.02%, raised 14DM mRNA and protein levels 2- to 3-fold. Addition of 2% Colestipol, a bile acid binding resin, to the chow diet caused smaller increases. The highest level of 14DM protein expression was observed in liver, the major site of feedback regulation of HMG-CoA reductase by cholesterol. Taken together, these observations suggest a critical role for 14DM in the feedback regulation of hepatic HMG-CoA reductase.
Assuntos
Anticolesterolemiantes/farmacologia , Colesterol/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/química , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , Oxirredutases/biossíntese , Oxirredutases/química , Animais , Western Blotting , Colestipol/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Immunoblotting , Lovastatina/farmacologia , Masculino , Modelos Biológicos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Esterol 14-Desmetilase , Fatores de Tempo , Distribuição TecidualRESUMO
Extraction of ER and PgR with 10 mM molybdate in TEDG buffer greatly facilitated their solubilization and a much higher receptor value was observed. However, since molybdate also affects the hormone receptor binding reaction, the apparent receptor activity assayed with molybdate is different from the actual receptor activity normally assayed without molybdate. A correction factor obtained by assaying cytosol with and without adding molybdate can be used to convert the apparent value into the actual value. The mathematical evaluation involving two separate effects of molybdate on receptors is illustrated.