Induction Of Malic Enzyme Research Articles

Ergosteroids V: preparation and biological activity of various D-ring derivatives in the 7-oxo-dehydroepiandrosterone series.

Our previous finding that D-ring seco derivatives of dehydroepiandrosterone retained biologic activity (Reich et al., Steroids 1998;63:542-53) motivated us to synthesize and test a number of steroids in which the D-ring is retained but altered in various ways. Several new steroids were synthesized and characterized by (1)H and (13)C NMR spectroscopy. The availability of a number of closely related compounds allowed detailed (13)C chemical shift correlations. Using the induction of two thermogenic enzymes in rats, liver mitochondrial glycerophosphate dehydrogenase (GPDH) and cytosolic malic enzyme, as criteria of biologic activity some 30 compounds were assayed. Hydroxylation of dehydroepiandrosterone (DHEA) at the 16 alpha position was previously shown to diminish activity (Lardy et al., Steroids 1998;63:158-65); the corresponding 7-oxo compound is fully active. Hydroxylation at the 15 beta position of DHEA, 7-oxo-DHEA, or 16 alpha-hydroxy-7-oxo-DHEA greatly diminished the induction of GPDH but induction of malic enzyme was retained. Most 5,15 diene steroids tested had 2 weak, or no, ability to enhance the formation of GPDH but did increase malic enzyme.

Response of hepatic proteins to 3,5,3'-tri-iodo-L-thyronine in diabetic rats.

In order to study whether peripheral action of thyroid hormones is altered in insulin deficiency and to elucidate the biological consequences of alteration of the cytosolic 3,5,3'-tri-iodo-L-thyronine (T3) binding protein (CTBP), we measured malic enzyme, T3-responsive nuclear n protein, CTBP and nuclear thyroid hormone receptor in the liver and kidney of streptozotocin (STZ)-induced diabetic rats that were treated with or without insulin and/or a receptor-saturating dose of T3. The following results were obtained. 1. Induction of malic enzyme by T3 was apparently diminished in diabetic rats. However, supplementary injection of insulin enabled previously given T3 to take effect in diabetic rats. 2. T3-responsiveness of other hepatic proteins (n protein and CTBP) was not altered by insulin in diabetic rats. 3. The level of n protein was increased by insulin in diabetic rats in vivo and in perfused rat liver, indicating that the hepatic n protein is a novel insulin-responsive protein. T3 and insulin increased the level of n protein non-synergistically in diabetic rat liver. 4. Hepatic nuclear receptor levels were not altered in diabetic rats. 5. Hepatic CTBP levels were decreased in diabetic rats. This was not due to the toxic effect of STZ. Low CTBP level was only partially increased by insulin after 30 days of diabetic period. Renal CTBP levels were not altered in diabetic rats with or without insulin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal induction of malic enzyme in rat hepatocytes cultured on laminin-rich gels

The levels of malic-enzyme mRNA and activity were determined in primary cultures of adult rat hepatocytes maintained on either rat-tail collagen or a laminin-rich substratum. Cells plated on laminin-rich gels exhibited substantially improved patterns of albumin and malic-enzyme expression when compared with cells maintained on rat-tail collagen. Moreover, hepatocytes plated on the laminin-rich matrix displayed marked malic-enzyme inducibility in response to tri-iodothyronine and dichloroacetate, especially in the presence of insulin. However, Northern blot analysis revealed that the ratio of the amounts of the two major malic-enzyme mRNA species (2.0 and 3.1 kb) was reversed when compared with that found in the liver in vivo, the altered levels of these two species being closer to those found in non-hepatic tissues. These findings indicate that, although the hormonal responsiveness of isolated hepatocytes maintained on laminin-rich gels is markedly improved, and approaches the degree of induction demonstrated in the liver in vivo, the mechanisms of control differ, indicating a loss of liver-specific expression.

Involvement of diminution of glutathione, produced by deficiency of methionine in the diet, in the elevation of malic enzyme level in rat liver

Rats fed on a low protein diet show an increase in the specific activity of malic enzyme and a concomitant decrease of glutathione concentration. We have studied the effect on malic enzyme activity of supplementing of low protein diet with essential amino acids. Only when methionine was excluded from the diet did the specific activity of malic enzyme increase to the same extent as found in rats fed with low protein diet. Immunoprecipitation of malic enzyme indicated that specific activity changes are the result of changes in the amounts of enzyme. Under all dietary conditions studied, the increase in malic enzyme activity is associated with a decrease in the concentration of GSH. To evaluate the possible causative role of GSH in malic enzyme induction, the specific activity of malic enzyme was measured in rats treated with BSO, an inhibitor of GSH biosynthesis. The reuslts show that in BSO-treated rats the decrease of GSH levels is also accompanied by an increase in the activity of malic enzyme.

Effects of ageing on transcriptional and post‐transcriptional regulation of malic enzyme and glucose‐6‐phosphate dehydrogenase in rat liver

We previously found an age-dependent impairment of induction of lipogenic enzymes in rat liver [Iritani et al. (1981) Biochim. Biophys. Acta 665, 636-639]. Further, we have found that after refeeding a fat-free diet to fasted rats, increases in transcriptional rate, mRNA concentration and enzyme induction of hepatic malic enzyme and glucose-6-phosphate dehydrogenase were always lower in 18-month-old rats than in 1.5-month-old rats. In the young rats, the transcriptional rates reached the maximum level in 4 h and the mRNA reached maximum levels in 16 h. The peaks tended to delay in the older rats. The half-lives of the mRNAs were not significantly longer in the old than in the young animals. The incorporation of [3H]leucine into the enzyme proteins was also decreased roughly in proportion to the enzyme induction. The mRNA concentrations in the liver polysomes were roughly proportional to the total mRNA. Thus, no effects of ageing on mRNA stability or on the translational activity of the enzymes could be found. It is suggested that the age-dependent decreases of malic enzyme and glucose-6-phosphate dehydrogenase induction can be mainly ascribed to the transcriptional steps. Moreover, the transcriptional rate, mRNA concentration and induction of malic enzyme were increased by triiodothyronine treatment at a similar rate in both the young and old rats, but the absolute increments were lower in the old animals. The triiodothyronine response to malic enzyme induction also appeared to be primarily decreased at the transcription level.

Rapid stimulation of hepatic oxygen consumption by 3,5-di-iodo-L-thyronine.

Tri-iodothyronine (T3) and thyroxine (T4) as well as 3,5-di-iodothyronine (T2) stimulated O2 consumption by isolated perfused livers from hypothyroid rats at a concentration as low as 1 pM by about 30% within 90 min. Application of T2 resulted in a faster stimulation than with application of T3 or T4. Inhibition of iodothyronine monodeiodinase by propylthiouracil, thereby blocking the degradation of T4 to T3 and of T3 to T2, demonstrated that only T2 is the active hormone for the rapid stimulation of hepatic O2 consumption: T3 and T4 lost all of their stimulative activity, whereas T2 was as potent as in the absence of propylthiouracil. Perfusion experiments with thyroid-hormone analogues confirmed the specificity of the T2 effect. The nucleus is unlikely to contribute to the rapid T2 effect, as can be deduced from perfusion experiments with cycloheximide and lack of induction of malic enzyme by T2. In conclusion, a new scheme of regulation of mitochondrial activity is proposed: T2 acts rapidly and directly via a mitochondrial pathway, whereas T3 exerts its long-term action indirectly by induction of specific enzymes.

Precocious induction of malic enzyme by nutritional and hormonal factors in rat foetal hepatocyte primary cultures

Rat foetal hepatocytes in primary cultures were used as a model for the study of malic enzyme gene expression. Carbohydrates and glycolytic metabolites produced the precocious induction of the malic enzyme in foetal hepatocytes cultured in the absence of serum and hormones. Palmitate prevented this induction. Insulin and triiodothyronine produced a significant increase in the malic enzyme specific activity in all the conditions studied. A synergistic effect between the two hormones is observed only when high concentrations of glucose are present. Glucagon prevents partially the induction produced by insulin plus triiodothyronine. Both carbohydrate and hormonal inductions of malic enzyme activity are related to parallel increases in its expression, and are prevented by protein synthesis inhibitors.

Response of hepatic mitochondrial alpha-glycerophosphate dehydrogenase and malic enzyme to 3,5,3'-triiodothyronine in streptozotocin-diabetic rats.

Hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosolic malic enzyme (ME) response to a single injection of a receptor-saturating dose of T3 were measured 10, 20, and 30 days after diabetes induction, and compared with values in controls either fed ad libitum (C) or under a restricted diet (FR). An insulin-treated diabetic (D + I) group was also included. Basal enzyme levels as well as enzyme response to T3 injection were correlated with nuclear T3 content, maximal nuclear T3-binding capacity (MBC) and equilibrium association constant (Ka). Diabetes for 10, 20, and 30 days was associated with a progressive decrease in the MBC; the mean decrease was 17%, 50%, and 59%, respectively, from the corresponding C values. The MBC in FR animals did not change appreciably during the experimental period. Moreover, neither the decreased MBC in D groups nor MBC in C, FR, or D + I animals were influenced by T3 injection. The Ka values were comparable in all experimental groups. Specifically bound nuclear T3 was decreased within the experimental period between 33% and 73% in D rats and 6% and 39% in FR rats respect to C values. T3 injection raised the mean nuclear T3 content in all groups. However, at each time interval the mean values of the nuclear T3 in D groups was significantly lower than that in C, FR, or D + I groups after T3 injection. The basal alpha-GPD activity tended to be relatively stable during the experimental period in both D and FR rats, whereas ME activity in D and FR groups was decreased, respectively, 52-64% and 18-39% from C values. The response of both alpha-GPD and ME to T3 injection in FR rats was comparable to that of C groups. The alpha-GPD response to T3 in D rats was not different from that of C rats on days 10 and 20 of the experiment, but on day 30 it decreased by 26%. In contrast, the induction of ME by T3 was severely decreased (by 66-88% of C values) within the experimental diabetes period. Thus, the measurements made in FR rats excluded the possibility that the quantitative changes in the enzyme response to T3 in D rats were nutrition-dependent. The differences between the response of alpha-GPD and ME to T3 in D rats suggest that cellular factors play a role in inhibiting or increasing the response to a given concentration of the T3-receptor complex.

Transcriptional and posttranscriptional regulation of malic enzyme synthesis by insulin and triiodothyronine

To investigate the transcriptional and posttranscriptional regulation of malate dehydrogenase (EC 1.1.1.40) induction by insulin, the transcriptional rate, mRNA concentration and enzyme induction of malic enzyme were compared in livers of normal and diabetic rats fed a high-carbohydrate diet. When rats were fed the diet for 4 days, the enzyme induction and mRNA concentration in livers of diabetic rats were only about 10% and 39%, respectively, of the values of normal rats, and the transcriptional rate was about 64%. Insulin treatment restored the transcriptional rate and mRNA concentration in 8 h and the enzyme induction in 4 days. Thus, it is suggested that insulin is involved in malic enzyme transcription of the gene and also possibly in the translation of the cytoplasm. On the other hand, by giving triiodothyronine treatment, the transcriptional rate and mRNA concentration were increased about twice and the enzyme induction, about 10-times in the diabetic animals. Triiodothyronine appears to stimulate malic enzyme transcription and possibly post-transcriptional steps even at a very low insulin level.

Nuclear triiodothyronine receptor of human adipose tissue

l-Triiodothyronine induces malic enzyme in explants from human adipose tissue. Consequently, we looked for the presence of receptors for l-triiodothyronine in nuclei isolated from human adipose tissue. The binding of 125I-triiodothyronine by the nuclei was time- and temperature-dependent. At 37°C binding reached a steady state after 60 minutes. Dithiothreitol enhanced total binding and suppressed nonspecific binding. Scatchard analysis showed the presence of a single class of binding sites. The apparent association constant, K a, was 0.13 ± 0.03 × 10 10 M −1, the maximal binding capacity 2.20 ± 0.81 pmol/mg DNA (mean ± SD, n = 7) and the number of binding sites 8,000/nucleus. l-Triiodothyronine and d-triiodothyronine had equal affinity to the nuclear receptor; triiodothyroacetic acid had three times higher affinity. l- and d-thyroxine had 8% and 12%, respectively, and tetraiodothyroacetic acid had 19% affinity compared to that of l-triiodothyronine. Reverse triiodothyronine was a weak competitor. Digestion of nuclei with micrococcal nuclease abolished specific binding. These results show that nuclei from human white adipose tissue possess high affinity receptors for l-triiodothyronine, which are associated with nuclear chromatin. It is likely that induction of malic enzyme in human adipose tissue by l-triiodothyronine is mediated by the nuclear receptors.

Activity of thyroid hormone-inducible enzymes following treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) caused a depletion of serum thyroxine, but paradoxically did not change l-3,5,3′-triiodothyronine (T3) levels in serum of rats. The activities of the thyroid-regulated enzymes α-glycerol phosphate dehydrogenase (GPD) and malic enzyme (ME) were determined in livers of normal and thyroidectomized (THX) rats treated with 0.1 to 100 nmol TCDD/kg body weight. Mitochondrial GPD activity did not change significantly as a function of TCDD dose in either normal or THX rats. ME activity was induced by TCDD in a dose-dependent fashion, but only in non-THX animals. The absence of ME induction in THX rats treated with TCDD indicates that TCDD is not intrinsically thyromimetic. The dependence of ME induction on thyroid hormones is much like the thyroid-hormone-dependent, multihormonal induction of ME by insulin and glucocorticoids. However, TCDD had no additive or synergistic effects on induction of ME activity in THX rats fed T3. A 30% decrease in steady-state plasma T3 levels of T3-fed animals treated with TCDD relative to T3-fed controls suggested that T3 catabolism was more rapid in TCDD-treated rats than controls. Thus a thyroid-hormone-dependent, multihormonal interaction is suggested as the basis for induction of ME by TCDD, but a strictly T3-dependent process has not been ruled out.

Free fatty acid inhibition of the insulin induction of glucose-6-phosphate dehydrogenase in rat hepatocyte monolayers.

Rat hepatocytes in monolayer culture were utilized to determine if the decrease in glucose-6-phosphate dehydrogenase (G6PD) activity resulting from the ingestion of fat can be mimicked by the addition of fatty acids to a chemically, hormonally defined medium. G6PD activity in cultured hepatocytes was induced several-fold by insulin. Dexamethasone or T3 did not amplify the insulin induction of G6PD. Glucose alone increased G6PD activity in cultured hepatocytes from fasted donors by nearly 500%. Insulin in combination with glucose induced G6PD an additional two-fold. The increase in G6PD activity caused by glucose was greater in hepatocytes isolated from 72 hr-fasted rats as compared to fed donor rats. Such a response was reminiscent of the "overshoot" phenomenon in which G6PD activity is induced well above the normal level by fasting-refeeding rats a high glucose diet. Addition of linoleate to the medium resulted in a significant suppression of insulin's ability to induce G6PD, but linoleate had no effect on the induction of G6PD activity by glucose alone. A shift to the right in the insulin-response curve for the induction of G6PD also was detected for the induction of malic enzyme and acetyl-CoA carboxylase. Arachidonate (0.25 mM) was a significantly more effective inhibitor of the insulin action than linoleate was. Apparently rat hepatocytes in monolayer culture can be utilized as a model to investigate the molecular mechanism by which fatty acids inhibit the production of lipogenic enzymes. In part, this mechanism of fatty acid inhibition involves desensitization of hepatocytes to the lipogenic action of insulin.

Influence of diet on the transcriptional and post-transcriptional regulation of malic enzyme induction in the rat liver.

Fasted rats were refed a carbohydrate/protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate) to investigate, using a cDNA cloned in our laboratory, the regulatory mechanisms involved in hepatic malic enzyme induction. In the carbohydrate/protein diet, although the enzyme activity and the mRNA concentration of malic enzyme were increased about 7-fold above the levels in the fasted rat, the rate of transcription was increased only 2-fold. In the carbohydrate diet group the rate of transcription and the concentration of mRNA were increased to the levels in the carbohydrate/protein diet group, whereas the enzyme activity increased only to 60% of those levels. Protein appears to contribute to an increase in the translation of malic enzyme mRNA. In the protein diet group the transcriptional rate was not low, but the mRNA concentration was about half in comparison with the level of the carbohydrate/protein diet group. Further, dietary fat did not reduce the transcriptional activity, but reduced the mRNA concentration and the enzyme activity to half of the basal levels. Therefore, it is suggested that fat stimulates the degradation of the mRNA in liver cytosol, whereas carbohydrate tends to stabilize the mRNA. On the other hand 3,5,3'-triiodothyronine treatment increased the transcriptional activity by 1.5-2-fold above the basal values on all the diets and even on fasting. Thus, it is suggested that 3,5,3'-triiodothyronine increases the transcriptional activity of malic enzyme independently from nutritional regulation, while the nutrients are predominantly involved in the post-transcriptional steps.

Effects of aging on contributions of dietary fat and triiodothyronine treatment to lipogenic enzyme induction

Although lipogenic enzyme inductions are reduced by fat feeding, this reduction decreases with aging and is particularly detectable in the case of acetyl-CoA carboxylase and fatty acid synthetase activities. On the other hand, the fat-dependent reductions of malic enzyme and acetyl-CoA carboxylase were consistently relieved by triiodothyronine (T 3) treatment. The effects of T 3 treatment on these enzyme inductions were greater in 10-month-old rats than in 1-month-old rats, while the carbohydrate-dependent induction and the fat-dependent reduction of the enzymes decreased with aging. In these animals, alterations in malic enzyme mRNA translational activities were roughly in parallel to the enzyme activities. Therefore, the age-dependent alterations in effects of T 3 treatment and fat on malic enzyme induction do not appear to occur in post-translation.

Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high-carbohydrate diet

Glucokinase and NADP:malate dehydrogenase (malic enzyme) first appear in liver when rat pups are weaned from milk which is high in fat to lab chow which is high in carbohydrate. To examine the influence of diet during the early neonatal period, before developmental changes in the circulating concentrations of thyroid and adrenocortical hormones occur, high-carbohydrate formula (56% of calories from carbohydrate), isocaloric and isonitrogenous with rat milk, was intermittently infused via gastrostomy starting on the second day of life. Pups had no further access to their dams. Body weights attained by these pups were at least 90% of those attained by mother-fed pups, which served as controls. In artificially reared rats fed the high-carbohydrate formula, on Day 4, glucokinase and malic enzyme were 30 and 18% of adult activity, respectively; on Day 10, glucokinase and malic enzyme were 71 and 96% of adult activity, respectively. On Days 4 and 10 glucose-6-phosphate dehydrogenase was elevated four- to fivefold in pups fed the high-carbohydrate formula compared to mother-fed pups. A second isocaloric formula, with 22% of calories from carbohydrate but low in protein, resulted in intermediate levels of all three enzymes on Day 10. Pups fed the high-carbohydrate formula has plasma insulin concentrations four- to fivefold greater than mother-fed pups on both Days 4 and 10. Triiodothyronine administration (1 μg/g body wt) on Day 1 enhanced the induction of malic enzyme but not glucokinase on Day 4 in pups fed the high-carbohydrate formula. The results demonstrate that neonatal rat liver is competent to respond to high carbohydrate intake by induction of glucokinase and malic enzyme.

Stimulation of malic enzyme formation in hepatocyte culture by metabolites: Evidence favoring a nonglycolytic metabolite as the proximate induction signal

Recent studies have shown that the addition of increasing concentrations of glucose to the medium of primary adult rat hepatocyte cultures results in the progressive induction of malic enzyme. We have undertaken experiments to determine (1) whether metabolism of glucose was an essential prerequisite for such induction, and (2) whether a specific glycolytic intermediate could be shown to constitute the proximate carbohydrate signal triggering such induction. In line with these objectives we investigated the ability of various sugars and glycolytic metabolites to induce malic enzyme in this system and assessed the influence of insulin, glucagon, and thyroid hormone (triiodothyronine, T 3) on this process. Our results show that only those sugars capable of entering the cell and being metabolized induce malic enzyme (glucose, fructose, and galactose). The nonmetabolizable sugars 3-O-methylglucose and 2-deoxyglucose are ineffective. Incubation with 20 mmol/L lactate, pyruvate, dihydroxyacetone, or glycerol resulted in malic enzyme induction, whereas incubation with acetate, citrate, and α-ketoisocaproate was without effect. The induction by all sugars and metabolites required presence of insulin. As previously reported for glucose, addition of T 3, under all metabolic conditions, resulted in a constant 3.6-fold increase in the rate of malic enzyme induction and further supports the proposal that T 3 acts to multiply the effect of a common carbohydrate-generated signal. Glucagon administration led to a dose-dependent inhibition of the carbohydrate effect with a half-maximal effect and maximal effect at 2 and 100 nmol/L, respectively. None of the glycolytic metabolites tested could reverse the glucagon inhibition completely. Our results indicate that metabolism of glucose is essential for induction of malic enzyme and are most consistent with the view that the signal arises from the metabolism of pyruvate at a step before the formation of cytsolic acetyl-CoA. Since these processes are mitochondrial in location, it appears likely that this organelle is the site of origin of the inducing signal. Of some interest in this connection was the finding that dichloroacetate led to a major induction of malic enzyme, even in the absence of insulin and in the presence of glucagon. The ability of dichloroacetate to bypass the glucagon block thus further supports a nonglycolytic origin of the inducing signal.

Cloning of cDNA sequences for murine malic enzyme and the identification of aberrantly large malic enzyme mRNA in MOD-1 null mice.

Polysomes containing cytosolic malic enzyme mRNA and malic enzyme nascent chains were complexed with specific antibodies and purified by chromatography on protein A-Sepharose. When poly(A+) mRNA derived from the immunoselected polysomes was translated in vitro, full length malic enzyme (subunit Mr = 58,000) accounted for a significant fraction (approximately 20%) of the polypeptides synthesized. Double-stranded cDNA, synthesized using partially purified malic enzyme mRNA as a template, was inserted into pBR 322 and cloned. Twenty-five candidate malic enzyme cDNA clones were identified by differential hybridization. Four clones were studied further and each of these was shown to have malic enzyme cDNA sequences by hybrid-selected translation and specific immunoprecipitation. Plasmid pME1, which contains a 1400-base pair insert, hybridized to two mouse liver malic enzyme mRNAs with lengths of 2300 and 3500 bases. Similar analyses were performed on liver mRNAs isolated from MOD-1 mutant mice which lack cytosolic malic enzyme activity. These Northern blots disclosed a pair of aberrantly large malic enzyme mRNAs with lengths of 2800 and 4000 bases. Furthermore, anti-malic enzyme antibodies exclusively precipitated a polypeptide translation product with a Mr of 77,000 when MOD-1 mRNA was used to direct in vitro protein synthesis. Thus, it is possible that MOD-1 malic enzyme mRNA contains an additional polypeptide coding sequence. The translation of such a sequence might disrupt enzyme function and/or markedly decrease enzyme stability. The malic enzyme cDNA probe was also employed to demonstrate that the induction of malic enzyme in the livers of previously starved mice that were fed a high carbohydrate, fat-free diet was controlled pretranslationally by a parallel modulation of the malic enzyme mRNA concentration.

Reciprocal effects of epidermal growth factor on key lipogenic enzymes in primary cultures of adult rat hepatocytes. Induction of glucose-6-phosphate dehydrogenase and suppression of malic enzyme and lipogenesis.

In primary cultured hepatocytes of adult rats epidermal growth factor (EGF) caused 2- to 3-fold induction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P dehydrogenase) within 2 days. The effect of EGF was additive with a similar effect of insulin. The half-maximum dose of EGF for the induction was 1 ng/ml. Induction of this enzyme by these hormones was shown by immunotitration to be due to increase of the amount of enzyme. Furthermore, this increase in the amount of enzyme was found to result from increase of syntheses of mRNA and enzyme protein. In contrast, the induction of malic enzyme (EC 1.1.1.40, L-malate:NADP+) oxidoreductase) by insulin plus triiodothyronine was strongly suppressed by the concomitant addition of EGF. Induction of G6P dehydrogenase by EGF, like that by insulin, was not suppressed by either glucagon or dibutyryl cAMP, whereas that of malic enzyme was suppressed additively by EGF and dibutyryl cAMP. EGF also suppressed stimulation of lipogenesis by insulin, measured as incorporation of [1-14C]acetate into triglycerides and phospholipids. Another difference between the inductions of G6P dehydrogenase and malic enzyme was in their dependence on cell density; G6P dehydrogenase induction by insulin and EGF was high at low cell density (3 X 10(4) cells/cm2) and less at higher cell density (13 X 10(4) cells/cm2), whereas induction of malic enzyme was high at higher cell density and less at lower cell density. These results are consistent with the dual role of G6P dehydrogenase in lipogenesis in resting cells and in synthesis of nucleic acid in growing cells. Malic enzyme plays a role only for lipogenesis in mature hepatocytes.

Comparison of age-related decreases in the basal and carbohydrate inducible levels of lipogenic enzymes in adipose tissue and liver

An age-related decrease in the level of four lipogenic enzymes—malic enzyme (ME), fatty acid synthetase (FAS), glucose-6-phosphate dehydrogenase (G-6-PD), and 6-phosphogluconate dehydrogenase (6-PGD)—was compared in the adipose tissue and liver of rats. In adipose tissue the activity of ME per milligram of protein at one month exceeded that observed at one year by a factor of 27 or more. The bulk of the fall occurred between one and three months. Adipose FAS, G-6-PD, and 6-PGD showed a similar but less precipitous age-related decline. The extent of age-related changes in the adipose tissues consistently exceeded those observed in liver preparations from the same animal. For both fat and liver, administration of a high carbohydrate fat-free diet for four days also elicited a substantially greater incremental ME response in young rats than in old animals. The age-related changes in basal ME activity were proportional to the increments produced by dietary stimulation. Thus, as previously proposed by us for liver, the decrease in basal ME in adipose tissue may be the result of a reduced generation from dietary carbohydrate of a stimulus responsible for the induction of lipogenic enzymes. Additional experiments were performed in animals provided with 10% glucose in their drinking water and infused with high doses of insulin for 4 to 12 days. In the fat of older animals, such treatment resulted in the induction of adipose ME activity to levels almost equivalent to those achieved with similar treatment in younger animals. Whereas maximal levels of ME in the younger animals could be attained simply with the glucose in the drinking water, insulin administratin was required in the older animals. Presumably, the insulin response evoked by glucose in the younger animals elicited a maximal ME increment. In the older animals, however, the endogenous insulin response to glucose failed to achieve a comparable degree of ME induction in the fat. Since the concentrations of insulin attained in the older animals were equal to or greater than those attained in the younger rats, both in the basal and the stimulated state, the diminished ME response in the older animals can be considered to be a form of tissue insulin resistance. Diminished glucose metabolism in the adipocytes of older rats is well established even in the presence of normal plasma glucose concentrations. Therefore, the results suggest that the level of hepatic and adipose ME activity may be useful as an index of such changes.

Interrelationship of triiodothyronine concentration, metabolism, protein binding, and nuclear occupancy in the induction of malic enzyme by cultured adult rat hepatocytes.

We have previously demonstrated the utility of the cultured adult rat hepatocyte system for the study of the induction of malic enzyme by T3. Although such induction could be demonstrated with medium both containing and lacking serum proteins, we noted that an 11-fold higher initial free T3 concentration was required to achieve a given level of enzyme induction when serum was absent from the medium. Since it appeared possible that this phenomenon might be related to an accelerated rate of T3 metabolism in the absence of serum, we undertook a theoretical and experimental analysis to assess the effect of reversible serum binding on both the rate of T3 metabolism and the rate of malic enzyme generation. We developed a mathematical model based on the provisional assumption that the medium proteins act exclusively through the reversible binding of T3. This allowed us to predict a number of parameters of metabolism and distribution as a function of serial protein dilution. Thus, as predicted by the model, seria...