Cyclic adenosine 3′,5′-monophosphate and glucose stimulate thyroxine 5′-deiodinase type II in cultured mouse neuroblastoma cells

Abstract

Nutrient modulation increases mouse neuroblastoma (NB) T 4-5′-deiodinase II (T 4-5′-D II) activity. Carbohydrates are more potent than either amino acids or glycerol as nutrient sources. Glucose rapidly (2 to 4 hours) enhances NB enzyme activity and the response is dependent on new protein synthesis. The present study was performed to further characterize this glucose effect and explore its relationship to the cyclic adenosine monophosphate (cAMP) system in these cells. NB T 4-5′-D II activity reached a maximum level (sixfold) in response to glucose (10 mmol/L) at 16 hours and thereafter remained constant up to 22 hours before reverting back to basal level between 24 and 30 hours. This pattern of response allowed the performance of detailed studies on maximum glucose activated NB T 4-5′-D II under transient equilibrium conditions during the 16- to 22-hour period. Addition of dibutyryl cAMP (dbcAMP) (1 mmol/L) at this stage significantly increased enzyme activity (twofold at 2 hours and fourfold at 4 and 6 hours) compared with glucose alone. There was an additive response to dbcAMP under these maximum glucose-activated conditions. Nonactivated NB T 4-5′-D II showed a twofold response to dbcAMP (1 mmol/L) at 4 hours in a glucose-free medium. Under these conditions, glucose (10 mmol/L) also increased enzyme activity twofold. Combined studies with dbcAMP and glucose increased enzyme activity fourfold at 4 hours. Subsequent studies were performed with forskolin (10 μmol/L) and cholera toxin (1 nmol/L), modulators of endogenous cAMP. The maximum enzyme response (2.5-fold) to forskolin was noted at 6 hours, whereas the peak effect of cholera toxin (twofold) occurred at 4 hours. Puromycin (100 μmol/L) decreased enzyme activity in the glucose-activated NB cell preparations at 2 hours (to 30% of control) and at 6 hours (to 50% of control). Coincubations (pre- and postpuromycin) with dbcAMP did not alter the puromycin effect at 2 and 4 hours. Actinomycin-D (1 μmol/L) did not alter NB T 4-5′-D II activity in the glucose-activated cells at either 2 or 4 hours postaddition, but did block the dbcAMP-induced increase in enzyme activity at 4 hours. Thus, mouse neuroblastoma T 4-5′-D II is responsive to cAMP modulation. The enzyme response to dbcAMP is not dependent on glucose, but is mediated by new protein synthesis. The responses to dbcAMP and glucose are additive. Thus, the glucose stimulation of brain T 4-5′-D II is not dependent on an increase in intracellular cAMP. The additive effects of glucose and dbcAMP suggest different mechanisms for the enhancement of neuroblastoma T 4-5′-D II activity.