Hormonal regulation of lipogenic enzymes in chick embryo hepatocytes in culture. Thyroid hormone and glucagon regulate malic enzyme mRNA level at post-transcriptional steps.

Abstract

Mechanisms involved in stimulation of the synthesis of malic enzyme by insulin and triiodothyronine and in inhibition of synthesis by glucagon have been investigated by assessing levels and rates of synthesis of malic enzyme mRNA in chick embryo hepatocytes in culture. Insulin alone had no effect on the level of malic enzyme mRNA, whereas triiodothyronine by itself caused a 7-fold increase. Insulin plus triiodothyronine caused an 11-fold increase. Glucagon caused a 93% decrease in the accumulation of malic enzyme mRNA caused by insulin plus triiodothyronine. Although the relative changes in mRNA level are smaller in magnitude, they are qualitatively similar to the effects of these hormones on synthesis of malic enzyme, suggesting that control is exerted primarily at pretranslational steps. After addition of triiodothyronine, malic enzyme mRNA accumulated with sigmoidal kinetics, approaching a new steady state at 36-48 h after adding hormone. Puromycin, an inhibitor of protein synthesis, blocked the effect of triiodothyronine if added 30 min prior to the hormone and inhibited further accumulation of malic enzyme mRNA if added 24 h after triiodothyronine. However, puromycin had no effect on the level of beta-tubulin mRNA (t1/2 = 3-5 h), suggesting that the effect of triiodothyronine on malic enzyme mRNA required synthesis of a peptide. Triiodothyronine increased transcription of the malic enzyme gene by 2-fold and level of its mRNA by 11-14-fold, indicating regulation is primarily at a post-transcriptional step. Glucagon caused malic enzyme mRNA to decay with a half-life of 1.5 h, whereas alpha-amanitin or actinomycin D, inhibitors of transcription, caused the mRNA to decay with a half-life of 8-11 h. The effect of glucagon was entirely post-transcriptional because the hormone had no effect on transcription. Taken together, these results suggest a model in which triiodothyronine regulates production of a peptide that stabilizes malic enzyme transcripts in the cytoplasm and/or nucleus. Glucagon may inhibit activity of the peptide induced by triiodothyronine.