Rat hepatic and renal 5′-deiodination of rT3 during fasting: Supportive role of intermediate Mr cytosolic non-glutathione thiol cofactor and NADPH

Abstract

The roles of subcellular components and, in particular, cytosol fractions and beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), in the regulation of rat hepatic and renal 5′-deiodination during fasting were assessed. 5′-deiodinase (5′-DI) activities in reaction mixtures were measured by using outer ring 125I-radiolabelled reverse triiodothyronine (rT3) as a substrate in the presence of 200 μmol/L NADPH. Subcellular components from rats fed ad libitum or fasted for 24, 48 or 72 hours were prepared by standard differential centrifugation. Cytosol was chromatographed on a Sephadex G-50 column to obtain Fraction A of molecular weight (Mr) >60,000 and Fraction B of Mr approximately 13,000 and to exclude reduced glutathione (GSH) (Mr<400). 5′-DI activity in liver homogenates was reduced by 42% at 24 hours and by 59% at 48 hours of fasting. In reconstitution experiments, liver microsomes showed a progressive loss of 5′-DI activity, reaching a maximal reduction of 48% at 72 hours of fasting. Activation of microsomal deiodinase by whole liver cytosol was also significantly reduced at 24 hours of fasting and achieved a maximal reduction of 5′-DI activation of 42% at 48 hours before substantial but incomplete recovery at 72 hours. Cytosolic Fraction A and B were assessed in combination with fed microsomes and NADPH. A close correlation was demonstrated between the loss of hepatic 5′-DI supportive activity in whole cytosol and that of Fraction B but not A during fasting. Furthermore, the changes observed in supportive activity in the intermediate Mr cytosolic Fraction B during fasting were found to parallel changes in the amounts of 14C-labelled product obtained by alkylation with 14C-iodoacetamide following reduction with NADPH and Fraction A. While the addition of NADPH at concentrations ranging from 1 nmol/L to 1 mmol/L further augmented hepatic 5′-deiodination in both fed and fasted preparations, it could not eliminate the observed differences in maximal 5′-DI activation between fasted and fed rats. In comparison with the changes in liver, there was little alteration in renal subcellular components including cytosolic fractions during fasting. These data obtained in the rat suggest that a non-glutathione, NADPH-dependent cytosolic dithiol generating system could have a physiologically important role in mediating microsomal 5′-DI activation, and that the change in hepatic 5′-deiodination rate may be due to altered availability of a cytosolic dithiol.