Thyroxine 5'-monodeiodinase activity in microsomes from isolated hepatocytes of rainbow trout: effects of growth hormone and 3,5,3'-triiodo-L-thyronine

Abstract

Rainbow trout hepatocytes isolated by collagenase perfusion were suspended in primary culture for up to 72 hr at 11 degrees and then the microsomal L-thyroxine (T4) 5'-monodeiodinase (5'D) activity was evaluated by 125I- generation from [125I]T4. The 5'D activity and Vmax (level of functional enzyme) and Km (Michaelis-Menten constant) values for microsomes obtained from incubated hepatocytes corresponded to those for microsomes obtained directly from intact livers. HPLC analysis revealed 3,5-[125I]3'-triiodo-L-thyronine (T3) as the only significant 125I-labeled organic product. Hepatocyte survival ( > 90%) and 5'D activity were unaltered by insulin (10(-9) M) in the incubate, but 5'D activity was inhibited by 10% fetal calf serum. Human growth hormone (hGH) at concentrations of 5-250 ng/ml did not increase 5'D activity. These results do not support previous in vivo studies demonstrating hGH-enhanced hepatic 5'D function in trout and indicate that either hGH acts indirectly on the liver to enhance 5'D activity or incubated hepatocytes lose GH responsiveness. However, coincubation of hepatocytes with T3 (15 or 30 nM) for 24 hr inhibited 5'D activity in a dose-dependent manner and induced the production of 3-[125I]3',5'-triiodo-L-thyronine (reverse T3). These data support previous in vivo studies in showing that T3 autoregulates its own hepatic production and show that T3 does so by acting directly on the hepatocyte to modify deiodination pathways.