Regulation of Malic Enzyme Synthesis by Insulin, Triiodothyronine, and Glucagon in Liver Cells in Culture*

Cells isolated from the livers of 17. to 19-day-old chick embryos were maintained in a chemically defined culture medium. During 3 days in culture the activity of malic enzyme, a measure of its concentration, was stimulated 2-, 23., or 77-fold by insulin, triiodothyronine, or insulin plus triiodothyronine, respectively. Glucagon blocked the stimulation caused by insulin plus triiodothyronine. Changes in the relative synthesis of immunologically isolated malic enzyme were similar in magnitude and direction to the changes in enzyme activity. Degradation of malic enzyme was unaffected by the three hormones. Both soluble protein and malic enzyme were degraded with a t, of about 30 hours. In cells preincubated for 2 days with synthesis of malic enzyme was stimulated within 3 hours after triiodothyronine and reached an apparent new steady state after to the of


Regulation of Malic
were collected by centrifugation and washed twice with ice cold 150 rn~ NaCl/lO rn~ oL-leucine/0.5% Triton X-100. The washed immunoprecipitates were dissociated in 1% sodium dodecyl sulfate and 1% P-mercaptoethanol (100" for 5 min) and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (8,9). After electrophoresis.
the gels were frozen and sliced. The slices were dissolved by heating overnight at 60" in 0.5 ml of 30% H,O,. Scintillation fluid (6 ml, Ref. 10) was added and the samples assayed for radioactivity.
Only radioactivity above background in the malic enzyme region of the gel was considered to have been incorporated into malic enzyme. The preparation and specificity of this antibody against malic enzyme has been described (3,8 after about a l-day lag period (Fig. 1).  nine resulted in a significant increase in the activity of malic enzyme within 7 hours (Fig. 3C). Rapid accumulation occurred during the next 40 hours (Fig. 3C; Ref. 3). Twenty-four hours after adding triiodothyronine, malic enzyme had increased to 12 times the activity at zero time and 8 times that of the control (Fig. 3C). Total protein mass was unchanged during the experiment (Fig. 3A). Relative synthesis of malic enzyme was significantly increased within. 3 hours after adding triiodothyronine (Fig. 30).
When expressed as a percentage of the synthesis at 24 hours (Fig.  3), the 3.hour value showed considerable variability. However, relative synthesis at 3 hours was 4.5 + 0.6 (mean * S.E.M., N = 5) times relative synthesis of the enzyme at zero time (p < 0.01). The increase caused by triiodothyronine was approximately linear for 24 hours (Fig.  30), after which it appeared to reach a plateau (Fig. 40). Twenty-four hours after adding triiodothyronine, relative synthesis of malic enzyme was 36 times that at zero time and 20 times that of the control cells (Fig. 30). Incorporation of leucine into total protein was not significantly stimulated by triiodothyronine (Fig. 3B). Time Course for Response to Glucugon-In cells preinduced with triiodothyronine, the subsequent addition of glucagon had no effect on enzyme activity within the first 7 hours (Fig. 4C). This result is consistent with the relatively long half-life of malic enzyme and the lack of effect ofglucagon on degradation. However, relative synthesis of the enzyme was only 73% of its respective control at 3.5 hours after adding glucagon (p < 0.025) and 29% at 6.5 hours (p < 0.001) (Fig. 40). Neither the mass of total cellular protein nor incorporation of leucine into soluble protein was affected by glucagon (Fig. 4, A  the plasma that communicates the nutritional state of the whole animal to the liver. Glucagon also regulates the synthesis of two other lipogenic enzymes; glucose-6-phosphate dehydrogenase (14) and fatty acid synthetase (15 glucagon regulates the synthesis of fatty acids on a minute to minute basis by regulating the activity of pre-existing enzyme molecules (16-H). All of these effects of glucagon appear to be mediated by cyclic AMP (1,14,15,17,19,20). The mechanisms whereby glucagon and cyclic AMP coordinate the regulation of the synthesis of the various lipogenic enzymes and the regulation of the activity of pre-existing lipogenic enzymes is the subject of continuing investigation.
A role for insulin in the specific regulation of malic enzyme synthesis in. uiuo is not supported in the experiments described here. Relative to the magnitude of the glucagon effect, the augmentation of enzyme synthesis caused by insulin was small, especially at lower, more physiological concentrations. The most pronounced effect of insulin alone was stimulation of the mass of total protein per plate. Insulin stimulates growth and other general cell functions in several cell types (21)(22)(23)(24). A similar general stimulatory effect may account for the effect of insulin on malic enzyme synthesis in liver cells in culture. Whether general in nature or not, the stimulation of total protein mass caused by insulin was probably not mediated by cyclic AMP because total protein mass was not reduced by glucagon.
Despite a recent report that chronic starvation lowers the plasma level of triiodothyronine (25), other evidence indicates that thyroid hormone does not play a major role in the regulation of the synthesis of the lipogenic enzymes during starvation and starvation followed by refeeding. Both hypoand hyperthyroid rats show typical, although somewhat muted, responses to starvation and starvation followed by refeeding (4,(26)(27)(28). Thyroid hormone in viuo'stimulates the total activities of several enzymes involved in lipogenic and nonlipogenie pathways (29). Hence thyroid hormone may play a permissive role, being required for the synthesis of several enzymes of diverse pathways.
The stimulation of malic enzyme synthesis caused by triiodothyronine was blocked by cu-amanitin (30), a specific inhibitor of eukaryotic RNA polymerase II, the enzyme respon- After 2 days in the presence of insulin, the medium was changed and the cells incubated for 24 hours in medium containing insulin plus triiodothyronine. Then, the medium was changed again to contain either insulin plus triiodothyronine (0) or the same medium plus glucagon (0