Interrelationship of Triiodothyronine Concentration, Metabolism, Protein Binding, and Nuclear Occupancy in the Induction of Malic Enzyme by Cultured Adult Rat Hepatocytes*

Abstract

We have previously demonstrated the utility of the cultured adult rat hepatocyte system for the study of the induction of malic enzyme by T₃. Although such induction could be demonstrated with medium both containing and lacking serum proteins, we noted that an 11-fold higher initial free T₃ concentration was required to achieve a given level of enzyme induction when serum was absent from the medium. Since it appeared possible that this phenomenon might be related to an accelerated rate of T₃ metabolism in the absence of serum, we undertook a theoretical and experimental analysis to assess the effect of reversible serum binding on both the rate of T₃ metabolism and the rate of malic enzyme generation. We developed a mathematical model based on the provisional assumption that the medium proteins act exclusively through the reversible binding of T₃. This allowed us to predict a number of parameters of metabolism and distribution as a function of serial protein dilution. Thus, as predicted by the model, serial dilution was associated with a linear increase in fractional turnover. In the presence of 10% calf serum, the t_½; of T₃ was 17 h; with dilution of serum to 5%, 2.5%, and 0%, the t_½; of T₃fell progressively to 14.2, 7.2, and 6.7 h. The theoretical analysis also appeared to provided insights into the results of dose-response studies. We observed that the total T₃ concentration required for achieving half-maximal effects was 1 nM, and the concentration required for maximum response was 500 nM T₃. Curiously, these concentrations appeared to be the same in the presence and absence of serum. Nevertheless, calculations based on the known nuclear affinity constant for T₃ predicted that the mean nuclear occupancy should be relatively independent of the serum content in medium. If the induction of malic enzyme is directly related to nuclear occupancy, this would provide an explanation of the dose-response relationships. The effect of serum in lowering the free hormone concentration of T₃ appears to be balanced by a slower fractional removal rate. Our results emphasize the importance of plasma protein binding and hormone metabolism in determining the biological effect of a given concentration of T₃. Our findings provide further support for the physiological relevance of the cultured hepatocyte model and afford a theoretical basis for studying the effect of plasma protein binding on the turnover and biological effect of other hormones in tissue culture systems.