Quantitation of rat liver messenger ribonucleic acid for malic enzyme

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Biochemistry 1981, 20, 3486-3492

Quantitation of Rat Liver Messenger Ribonucleic Acid for Malic Enzyme during Induction by Thyroid Hormonet Howard C. Towle,* Cary N. Mariash, Harold L. Schwartz, and Jack H. Oppenheimer

ABSTRACT: The induction of cytosolic malic enzyme in rat liver

by 3,5,3’-triiodo-~-thyronine (TJ is due to an increase in the level of messenger ribonucleic acid (mRNA) coding for this protein. To investigate the mechanism of this hormone action, we measured the doseresponse relationship between T3and malic enzyme mRNA and the kinetics of appearance and disappearance of malic enzyme mRNA following T3 administration and withdrawal, respectively. Messenger RNA coding for malic enzyme was quantitated by using the mRNA-dependent rabbit reticulocyte lysate translational system. Identification of malic enzyme in the translational products was achieved by chromatography on N6-(6-aminohexy1)adenosine 2’,5‘-diphosphate-agarose, a biospecific affinity resin for NADP-dependent enzymes, followed by specific immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this procedure, levels of malic enzyme mRNA as low as 0.003% of the total translational products in normal, uninduced rat liver could be measured.

R a t liver malic enzyme [L-malate:NADP+ oxidoreductase (decarboxylating), EC 1. l a1.401 responds markedly to the thyroidal status of the animal (Tepperman & Tepperman, 1964; Wise & Ball, 1964). Thus, the transition from the hypothyroid to the extremely hyperthyroid state results in a 20-30-fold increase in malic enzyme activity (Oppenheimer et al., 1977). The increase in enzyme activity has been shown to result from an increased rate of malic enzyme synthesis, leading to an induction in enzyme mass (Isohashi et al., 1971; Silpanata & Goodridge, 1971; Gibson et al., 1972; Murphy & Walker, 1974; Li et al., 1975). Recently, we have measured the level of hepatic messenger ribonucleic acid (mRNA)’ coding for malic enzyme (Towle et al., 1980). Experimental hyperthyroidism induced by the injection of 15 pg of triiodothyronine (T3)l (100 g of body weight)-’ (day)-’ for 7 days led to proportional increases in mRNA and enzyme activity for malic enzyme. Thus, the induction of malic enzyme by thyroid hormone was mediated by an increase in the cellular content of specific mRNA. To further elucidate the mechanism of induction of malic enzyme mRNA following T3 administration, we have attempted to more closely define the response characteristics of this system. For this purpose, it was necessary to develop a more sensitive assay technique for detecting malic enzyme mRNA. This paper describes this technique, as well as the kinetics and dose-response relationship of the mRNA induction process. In addition, our studies allow us to prepare a mathematical model describing the relationship of malic enzyme to its mRNA at full receptor occupancy. ‘From the Department of Biochemistry and the Division of Endocrinology and Metabolism, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455. Received August 26, 1980. This work was supported by grants from the National Institutes of Health (AM-26919 and AM-19812) and Clinical Investigator Award AM00800 (C.N.M.).

0006-2960/8 1/0420-3486$01.25/0

Treatment of euthyroid rats with varying doses of T3 led to proportional increases in both malic enzyme activity and specific mRNA coding for malic enzyme, indicating that hormonal action is due entirely to changes at a pretranslational level. Following acute administration of T3 to normal rats, a lag time of approximately 2-3 h was found prior to the earliest effect on malic enzyme mRNA level. From the kinetics of mRNA appearance, a half-time of 12 h was estimated for malic enzyme mRNA in the presence of hormone. A similar half-time of 10 h was determined following withdrawal of animals from T3 treatment. T3 did not appear to act by stabilizing malic enzyme mRNA and, thus, must be acting at some step leading to increased production of mRNA. Based on the half-times of malic enzyme and its mRNA, a mathematical model of enzyme accumulation following T3induction was derived. This model demonstrates that no significant refractory period exists prior to the initiation of malic enzyme induction by T3.

Experimental Procedures

Animals. Male Sprague-Dawley rats weighing 200-250 g were used in all experiments. Animals were fed standard chow diet (Ralston-Purina) ad libitum and maintained on a 12-h light-12-h dark cycle. T3 (Sigma Chemical Co.) was dissolved in a minimum volume of 0.1 N NaOH, diluted to the appropriate concentration in standard saline, and injected intraperitoneally at the indicated doses. Animals were killed by exsanguination. Livers were rapidly removed, stripped of connective tissue, and rinsed in cold saline. A 2-g portion of liver was removed for determination of enzyme activity. The remainder was quickly frozen in liquid N 2 and stored at -80 OC until RNA extraction. Malic Enzyme Assay. Freshly excised liver was homogenized in 9 volumes of 0.32 M sucrose, 3 mM MgClz, and 10 mM Tris-HC1, pH 7.6, and a sample was removed for DNA analysis (Giles & Myers, 1965). Homogenates were centrifuged at 130000g,, for 1 h at 4 OC. The supernatant fraction (cytosol) was assayed for malic enzyme by the method of Hsu & Lardy (1967). One unit of enzyme activity catalyzed the reduction of 1 nmol of NADPf in 1 min. Total protein was measured by the method of Lowry et al. (1 95 1) using bovine serum albumin as a standard. Purification of malic enzyme and preparation of antibody to the purified enzyme were as previously described (Towle et al., 1980). The IgG fraction of serum was used in all ~~

Abbreviations used: mRNA, messenger ribonucleic acid; DNA deoxyribonucleic acid; T3, 3,5,3’-triiodo-~-thyronine; Sac, heat-killed, formaldehyde-fixed Staphylococcus aureus (Cowens I strain); NaDodSO4, sodium dodecyl sulfate; Tris, tris(hydroxymethy1)aminomethane; poly(A), poly(adeny1ic acid); Hepes, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid; ATP, adenosine 5’-triphosphate; GTP, guanosine 5’-triphosphate;EDTA, ethylenediaminetetraaceticacid; TSH, thyroidstimulating hormone.

0 1981 American Chemical Society

experiments. 3H- or 14C-labeledmalic enzyme was prepared by the formaldehyde labeling procedure of Rice & Means (1971). Isolation of Poly(A)-ContainingRNA. Total cellular RNA was isolated from frozen liver samples by extraction with phenol-hloroform at pH 9 as described (Towle et al., 1980). Poly(A)-containing RNA was obtained from total cellular R N A by chromatography on oligo(dT)-cellulose (Collaborative Research). In Vitro Translational Assay. Translational assays were run by using the micrococcal nuclease treated rabbit reticulocyte lysate system (Pelham & Jackson, 1976). Treatment of lysate with 60 pg/mL nuclease (P-L Biochemicals) for 12 min at 20 OC led to a reduction in (-)RNA controls to 0.2-0.5% of untreated lysate. Addition of rat liver poly(A)-containing RNA led to stimulations of 15-20-fold over the (-)RNA backgrounds. Translational assays contained in 120-360 pL of volume 20 mM Hepes, pH 7.6,80 mM KC1, 2 mM magnesium acetate, 1 mM ATP, 0.2 mM GTP, 15 mM creatine phosphate, 40 pg/mL creatine phosphokinase, 80 pM 19 unlabeled amino acids, and either 20 pCi of [3H]leucine (120 Ci/mmol) or 67 pCi of [3SS]methionine(700 Ci/mmol) (New England Nuclear). Incubation was for 90 min at 23 OC. Following incubation, reactions were centrifuged at 13OOOOg,, for 1 h at 2 OC, and the supernatant fraction was used for further analysis. In samples in which recovery was monitored, lo4 cpm (-2 pg) of 3H-labeled malic enzyme (for [3sS]methionineincorporation) or 14C-labeledmalic enzyme (for [3H]leucineincorporation) was added to the reaction prior to centrifugation. Quantitation of Malic Enzyme in Translational Products. To each sample was added 200 pL of a 25% (v/v) suspension of N6-(6-aminohexyl)adenosine 2’,5’-diphosphate-agarose in 20 mM Tris-HC1, pH 7.6, 0.2 mM EDTA, and 1 mM dithiothreitol (buffer A). After incubation for 60 min at 4 OC, the affinity resin was collected by centrifugation for 2 min in a Beckman microfuge. The supernatant was removed by aspiration and the resin was washed 4 times by resuspension in 500 pL of buffer A. After being washed, malic enzyme was eluted in 150 pL of 0.5 mM NADP+ in buffer A ( 5 min at 4 “C). The eluted products were separated from the resin by centrifugation and then adjusted to 0.25% (v/v) NP-40. Samples were then further purified by immunoadsorption with specific antibody to malic enzyme and with heat-killed, formaldehyde-fixed Staphylococcus aureus cell wall preparation (Sac, Calbiochem), as previously described (Towle et al., 1980). Following NaDodS04-polyacrylamide gel electrophoresis on 7.5% gels, radioactivity comigrating with purified malic enzyme was quantitated, corrected for recovery, and expressed as a percentage of the total incorporation. Results Detection of Malic Enzyme mRNA Activity in Uninduced Animals. We have previously demonstrated that malic enzyme mRNA activity could be detected and quantitated in hepatic poly(A)-containing RNA isolated from hyperthyroid rats. For this purpose, RNA was translated in the nuclease-treated reticulocyte lysate system, and radiolabeled products were analyzed for malic enzyme by using specific antibody to the purified protein. Antigen-antibody complexes were collected by using S a c as an immunoadsorbent. With [35S]methionine as the radiolabeled precursor, -0.04% of the products translated from hepatic RNA were found as malic enzyme in these induced animals (Towle et al., 1980). Efforts to quantitate malic enzyme mRNA from livers of normal, uninduced rats were largely unsuccessful. Since the

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VOL. 20, NO. 12, 1981

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1: Translation of malic enzyme from euthyroid rat liver. Poly(A)-containing RNA was isolated from the liver of a euthyroid rat and translated in an assay of 360 pL by using [’%]methionine. Products were analyzed by the combined affinity resin-immunoprecipitation procedure. Incorporation into newly synthesized polypeptides was 6 X lo5 cpm. A control reaction was run in parallel in which nonimmune rabbit IgG replaced anti-malic enzyme IgG during the immunoadsorption (0). FIGURE

RNA isolated from normal rats was not defective in its overall translational activity, we presumed that this difficulty reflected the extremely low levels of malic enzyme mRNA present in uninduced animals. Attempts to increase the synthesis of malic enzyme by scaling up the translational assay were of little value, since no enhancement of the signal to noise ratio was obtained. Therefore, a further purification step was added to the detection procedure. The biospecific affinity resin, M (6-aminohexy1)adenosine 2’,5’-diphosphateagarose, which had previously been used by Goodridge et al. (1979) for a similar purpose, was chosen. Only a limited number of NADP+-dependent proteins, including malic enzyme, are capable of binding to this resin (Brodelius et al., 1974). Use of the affinity resin prior to immunoprecipitation greatly reduced (> 10-fold) levels of nonspecific background with no substantial reduction in the level of specific malic enzyme (data not shown). Although the recovery of malic enzyme in each of the steps used for detection is quite high (>70%), potential variations could adversely affect attempts to quantitate malic enzyme mRNA activity. Therefore, the procedure has been adapted by addition of an internal standard for estimating recovery. When [3sS]methioninewas used as precursor, 3H-labeledmalic enzyme was added to the translational products after incubation. NaDodS04-polyacrylamide gels were simultaneously counted for 3H and 35S. The recovery of 3H-labeled malic enzyme was used to correct the amount of 35S-labeledmalic enzyme found in the translational products. The mean recovery of malic enzyme by the combined affinity resin-immunoadsorption procedure was 51.9% (n = 12) with a standard deviation of 9.6% (coefficient of variation = 18.5%). Intraassay variability was slightly less with a coefficient of variation of 9.4%. Using the improved detection procedure for malic enzyme, we attempted to translate hepatic poly(A)-containing RNA from the normal animal. The results are shown in Figure 1, A peak of radioactivity comigrating with malic enzyme was detected. No peak was observed when preimmune rabbit sera replaced anti-malic enzyme IgG in the immunoadsorption. In this assay, 6 X lo6 cpm of [3SS]methioninewas incorporated into total protein. Backgrounds were reduced to