Induction Of Malic Enzyme Research Articles

Glucose and triiodothyronine both induce malic enzyme in the rat hepatocyte culture: evidence that triiodothyronine multiplies a primary glucose-generated signal.

We have stimulated in a cultured hepatocyte system the synergistic interaction between triiodothyronine (T3) and dietary carbohydrate in the induction of malic enzyme (ME). Kinetic studies revealed that isolated hepatocytes equilibrate with media T3 within 5 min; nuclei equilibrate with media T3 by 45 min; and the half-time of T3 metabolism was 10 h in 10% serum. We demonstrated nuclear T3 receptors in isolated hepatocytes and the induction of ME by T3 in physiological concentrations. However, in the complete absence of T3 glucose could still induce ME. At all concentrations of glucose (100-1,000 mg/dl), T3 (0.3 nM free T3) resulted in a relatively constant (1.4- to 1.7-fold) increase in ME response. The augmentation in ME activity was paralleled by an enhanced rate of enzyme synthesis as determined by [3H]leucine incorporation into immunoprecipitable ME. Cells cultured in serum free media also demonstrated a glucose-dependent increase in ME. Insulin greatly stimulated the glucose induction of ME, whereas dexamethasone had very little influence on ME induction. These studies demonstrate the usefulness of an adult hepatocyte tissue culture model for the study of the effects of T3 on gene expression in cells that are not derived from tumor. They clearly demonstrate that well established effects of T3 can be simulated in such a system at levels of free hormone that approximate those in extracellular body fluids. Our results indicate that an increased concentration of glucose per se can induce the formation of ME in the absence of alterations in extrahepatic hormones or factors. Moreover, our findings confirm inferences from in vivo studies that T3 acts as a multiplier of a glucose-induced signal.

Inhibition of malic enzyme induction by triiodothyronine in the diabetic rat: Reversal by fructose feeding

In order to determine whether an increase in insulin is a prerequisite for induction of malic enzyme (ME) by triiodothyronine (T 3), we examined the ability of various diets to affect the T 3 induction of ME in diabetic rats. Forty-eight hours after streptozotocin, rats were placed on either a regular diet, a 60% glucose diet, or a 60% fructose diet. The next day selected groups received 200 μg T 3/100 g body weight. Twenty-four hours later serum was collected and the liver 100,000 × g supernatant fraction was assayed for ME. Compared to intact controls, the level of ME in the basal state as well as in the T 3 stimulated state was significantly less in diabetic rats (in U/mg, basal: intact, 19.3; diabetic, 7.1; T 3-treated:intact, 56.6; diabetic, 15.6). Fructose feeding in the diabetic rats led to no significant increment in ME (1.1 U/mg) whereas the intact rat fed fructose showed a significant increment in ME (39.4 U/mg). The combined administration of fructose and T 3 in the diabetic rat however, resulted in a substantial increment of ME (30 U/mg), which was significantly greater than the increment induced by T 3 and glucose (2 U/mg). Moreover, the increment in ME induced by T 3 and fructose in diabetic animals was not significantly different from that achieved in intact animals (54.9 U/mg). All groups of diabetic rats showed a decrease in insulin from the start of the dietary manipulations to the end of the experiment (28.7 μU/ml fasting to 10.9 μU/ml when killed in the fed state). Thus ME induction by T 3 can occur in the absence of an increase in insulin secretion. Fructose feeding can supply, at least in part, the necessary factors that permit T 3 to induce ME in diabetic rats.

Synergism of thyroid hormone and high carbohydrate diet in the induction of lipogenic enzymes in the rat. Mechanisms and implications.

We have investigated the relationship between the administration of triiodothyronine (T3) and a high carbohydrate (CHO) fat-free diet in the induction of lipogenic enzymes in two rat tissues, liver, and fat. Male thyroidectomized rats were treated with graded daily doses of T3 and either supplemented with a high CHO diet or left on a regular diet. Enzymes studied included malic enzyme (ME), fatty acid synthetase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. In the liver, all four lipogenic enzymes showed a synergistic response between T3 administration and high CHO feeding. In fat, ME also responded synergistically. The interaction was reflected in an increased sensitivity to T3. The dose of T3 required to achieve 50% maximal response was reduced three- to seven-fold by the high CHO diet. This phenomenon could not be attributed to a dietary-induced alteration either in T3 metabolism or in number or affinity of the T3-nuclear receptors. Moreover, studies of the relative rate of synthesis of ME suggested a simultaneous time of onset in the induction of ME, within 2 h after the application of either T3 or CHO. Thus, it is unlikely that either stimulus is secondary to the other. Since parallel experiments from this laboratory (Towle, Mariash, and Oppenheimer,1980. Changes in hepatic levels of messenger ribonucleic acid for malic enzyme during induction by thyroid hormone or diet. Biochemistry. 19: 579-585.) show that ME induction both by CHO and T3 is mediated by an increase in specific messenger RNA for ME, the interaction of T3 and the dietary factor occurs at a pretanslational level.

Changes in the hepatic levels of messenger ribonucleic acid for malic enzyme during induction by thyroid hormone or diet.

Levels of hepatic messenger ribonucleic acid (mRNA) for malic enzyme [L-malate:NADP oxidoreductase (decarboxylating), EC 1.1.1.40] were quantitated in different dietary and hormonal states of the rat. Polysomal or total cellular poly(A)-containing RNA was translated in the rabbit reticulocyte lysate system, which had been treated to reduce endogenous mRNA activity. The relative level of incorporation of radiolabeled amino acid into malic enzyme was determined by immunoprecipitation with antibody to malic enzyme and formaldehyde-fixed Staphylococcus aureus (Cowens I strain) as an immunoadsorbent. The immunoprecipitated product comigrated with purified malic enzyme on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. No malic enzyme was detected when nonspecific antisera or an excess of unlabeled malic enzyme was added during immunoprecipitation. The level of malic enzyme mRNA was found to markedly increase relative to euthyroid, chow-fed rats when the animal was either fed a high carbohydrate, fat-free diet or made hyperthyroid. Animals receiving both treatments had a further increase in mRNA activity to a level which was approximately 0.2% of the total incorporation of [3H]leucine. Levels of malic enzyme activity and the relative rate of synthesis were found to increase roughly in proportion to mRNA levels in these three states. Thus, the induction of malic enzyme by thyroid hormone or high carbohydrate, fat-free diet is due largely to an increase in the mRNA coding for this enzyme.

Relationship between the Accumulation of Pituitary Growth Hormone and Nuclear Occupancy by Triiodothyronine in the Rat

Studies were undertaken in hypothyroid rats in an effort to define the kinetics of growth hormone (GH) accumulation in response to i.v. pulse injections of triiodothyronine (T(3)) and to calculate the relationship between nuclear occupancy by T(3) and the instantaneous rate of accumulation of pituitary GH. Results were contrasted to the findings in previous studies of the induction of hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and malic enzyme (ME) by T(3). The dose of T(3) required to achieve half-maximal accumulation of GH in 24 h was 0.6 mug/100 g body wt, a value 15-fold less than the half-maximal dose for alpha-GPD and ME induction at a comparable time after injection. Although significant increase in pituitary GH were evident as early as 3 h after injection of maximally effective doses of T(3), the rate of increase became linear only 12 h after injection. After achievement of peak values, the pituitary content of GH decayed with a similar terminal t((1/2)) of 3.9 days and 4.1 days in two groups of animals injected with a single dose of 1.0 and 50 mug T(3)/100 g body wt, respectively. In vivo isotopic displacement studies carried out at the equilibrium time point indicated that the pituitary nuclear binding capacity was 5.5 ng T(3)/g tissue and that the plasma concentration at which one-half of the nuclear sites are occupied is 1.0 ng/ml. Nuclear occupancy as a function of time was calculated from the estimated plasma T(3) concentration after injection of the dose and the half-occupancy plasma concentration. These data were then analyzed by application of the mathematical model previously developed to ascertain the relationship between nuclear occupancy and the rate of hepatic enzyme induction. Results indicated that the pituitary nuclear occupancy-response relationship was generally linear, in marked contrast to the highly amplified relationship between nuclear occupancy and the response of ME and alpha-GPD to T(3) in the liver. In supplementary experiments, euthyroid rats received daily injections of 200 mug of T(3) for 7 days to keep nuclear sites nearly saturated for the duration of the experiment. No significant increase in the pituitary GH content above euthyroid base-line levels was noted. This also contrasts with the marked increase above euthyroid levels in alpha-GPD and ME observed in previous studies. Our findings suggest the existence of major differences between the specific mechanisms which lead to the induction of pituitary GH and the hepatic enzymes by T(3).

Stimulation of hepatic mitochondrial alpha-glycerophosphate dehydrogenase and malic enzyme by L-triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated.

Experiments were designed to analyze the relationship of a single i.v. dose of triiodothyronine (T3), the level of plasma and hepatic nuclear T3 attained, and the tissue response as reflected in increased activity of hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosol "malic enzyme" (ME). These studied were carried out in euthyroid rats by varying the dose of T3 injected and the time at which the animals were killed and the enzyme levels measured. The plasma T3 concentration was determined and the fraction of nuclear sites occupied at any time t was calculated from the known plasma:nuclear relationship. As a first step, the analysis was confined to the limiting situation in which all nuclear sites were effectively saturated. The following additional information was required and obtained: A proportional relationship between the half-neutralizing volume of a specific antiserum to malic enzyme and the activity of malic enzyme was established, thus confirming previous reports that the increase in enzyme activity induced by T3 is due to increased enzyme mass. The absolute refractory period immediately after i.v. injection of T3, during which no enzyme response could be detected, was determined. This was shown to be 13.4 h for alpha-GPD and 8.2 h for ME. Lastly, the t1/2 of the enzyme decay after pulse injection of T3 was measured. This was similar for both enzymes, 2.8+/-0.6 (SD) days for alpha-GPD and 2.7+/-0.6 (SD) days for ME. The results of these studies indicated that the extent of hepatic response appears limited by full occupancy of a set of intracellular receptor sites by T3 which is in rapid equilibrium with the plasma hormone pool. The kinetic properties of the receptors, as functionally defined in these studies, resemble those associated with the recently described specific nuclear T3 sites. These data per se are thus compatible with but do not prove a nuclear site of initiation of hormone effect. Thye do allow the development of an internally consistent mathematical model which permits prediction of enzyme response when the receptor sites are fully occupied for a given length of time after the i.v. injection of hormone. A separate series of studies was carried out in thyroidectomized rats. The response characteristics of alpha-GPD were similar to those observed in euthyroid animals. In contrast, however, the early response of ME to pulse injections of T3 was very much reduced in hypothyroid animals as compared to euthryoid animals in which nuclear sites were saturated for comparable periods. These findings raise the possibility that a factor required for the induction of malic enzyme but not alpha-GPD is deficient in the hypothyroid state.

Malate utilization by a group D Streptococcus: regulation of malic enzyme synthesis by an inducible malate permease.

Induction of an nicotinamide adenine dinucleotide-specific malic enzyme and a malate entry system permits Streptococcus faecalis to grow at the expense of malate. Evidence is presented which shows that biosynthesis of the permease, but not of the malic enzyme, is subject to catabolite repression by glucose. In contrast to the malic enzyme, the catalytic function of the entry system does not appear to be inhibited by intermediate products of glycolysis. Although the induction of the entry system does not appear to be coordinated with the induction of the malic enzyme, the latter process is dependent upon the permease for the transport and accumulation of inducer.

Induction of malic enzyme and of oxalacetate decarboxylase in three lactic acid bacteria.

SUMMARY: The presence of inducible, DPN-specific malic enzymes have been demonstrated in Lactobacillus plantarum, L. casei and Streptococcus faecalis. Of 30 compounds tested, only l-malate induced the synthesis of malic enzyme in L. plantarum. The possibility that the malic enzymes of these three organisms are bifunctional proteins which have separate sites to decarboxylate oxalacetate has been excluded. In addition to l-malate, three compounds, oxalacetate, acetoacetate and α-keto- glutarate, induce the synthesis of oxalacetate decarboxylase even though they cannot induce the synthesis of malic enzyme. The oxalacetate decarboxylation system consists of an oxalacetate decarboxylase and an oxalacetate permease. Malate can induce the synthesis of only oxalacetate decarboxylase and not oxalacetate permease.

Malic enzyme induction by lactic acid bacteria. II. Purine and pyrimidine requirements.

Malic enzyme activity was shown to be inducible in cells of Lactobacillus arabinosus, Lactobacillus casei, Lactobacillus delbrueckii, Leuconostoc mesenteroides, and Streptococcus faecalis.Inspection of the nitrogen requirements for induction revealed an obligatory need for purines and uracil by L. casei and L. delbrueckii. Addition of purines and uracil also stimulated induction by S. faecalis and L. arabinosus, but not by L. mesenteroides. The role of purines was inspected further by the inhibitory action of 8-azaguanine.Studies on protein and RNA synthesis during malic enzyme induction revealed that RNA synthesis preceded protein synthesis. This phenomenon was masked in the absence of added purines and uracil by concomitant degradation of cellular RNA. Such breakdown of RNA was observed also when the cells were aged at pH 7.The stimulatory effect of biotin on malic enzyme induction by L. arabinosus could be replaced partially by adenine, and the inhibitory effect of homobiotin was reversed by the addition of this purine. The absence of a biotin effect on induction by L. casei may be related to the requirement for purines and uracil in this organism.

Malic enzyme induction by lactic acid bacteria. I. A method for the study of nutritional interactions.

The nutritional requirements for the induction of malic enzyme by washed cellular suspensions of Lactobacillus arabinosus 17-5 have been found to be quite similar to the growth requirements for this organism. This similarity permits the use of the measure of rate and extent of malic enzyme synthesis as a tool for studying amino acid interactions and vitamin function.