Nutritional and hormonal regulation of malic enzyme synthesis in rat mammary gland.

Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was constant during the first 6 h of starvation. When lactating rats (maintained with their pups) were starved for 24 h and then re-fed, the relative rate of enzyme synthesis increased 2.5-, 4-, and 4.5-fold at 3 h, 6 h and 18 h respectively after initiation of re-feeding. The relative rate of malic enzyme synthesis was about 50% of normal at 15 h after weaning, whereas the rate of synthesis of soluble proteins did not change. Administration of bromocriptine or adrenalectomy of lactating rats decreased the relative rate of synthesis of malic enzyme by 40% or 30% respectively; these effects were counteracted by hormone supplementation. Hormone therapy also caused an increase in the rate of incorporation of [3H]leucine into soluble proteins and in malic enzyme activity.     

Tissue-specific regulation of two functional malic enzyme mRNAs by triiodothyronine

Rat liver malic enzyme (ME) synthesis is known to be regulated by 3,5,3'-triiodo-L-thyronine (T3). Hybridization of 32P-labeled ME cDNA with RNA extracted from normal and T3-induced livers (15 or 50 micrograms/100 g body weight for 10 days) showed an increase in the ME mRNA concentration by approximately 11-fold in T3-treated rats. ME activity and ME mass were stimulated to the same degree as ME mRNA. Northern blot analysis of either total or poly(A+) RNA revealed two distinct ME mRNAs (21 and 27 S) which were equally induced by T3 treatment. Both mRNAs were shown by in vitro translation assay to program the synthesis of the same immunoprecipitable protein corresponding to full-sized ME. From all the above, we concluded that both messages code for active enzyme. ME activity and ME mRNA were also detected in nonhepatic tissues for which different responses to T3 induction were observed without direct correlation with their respective content of T3 nuclear receptor. Increases in ME activity and level of hybridizable ME mRNA were seen 48 h after a single administration of T3 (200 micrograms/100 g body weight) in liver, kidney, and heart (10.3- and 15.5-, 1.7- and 2.6-, and 1.72- and 3.4-fold above basal values, respectively). Lower levels of induction could already be detected after 24 h, liver being the most stimulated tissue. ME was not affected in brain, lung, testis, and spleen. Northern blot analysis showed that both ME mRNAs are present in all tissues tested, although in different relative proportions.(ABSTRACT TRUNCATED AT 250 WORDS)     

High malic enzyme activity in tumor cells and its cross-reaction with antipigeon liver malic enzyme serum

Summary Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserumin vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP⁺ orL-malate. Metal Mn²⁺ gave little protection.     

Thyrotropin regulation of malic enzyme in FRTL-5 rat thyroid cells

TSH-induced increases in malic enzyme mRNA levels in FRTL-5 rat thyroid cells are paralleled by increases in malic enzyme activity and are mimicked by 8-bromo-cAMP. Apparent approximately 4 h after TSH challenge and maximal after 16 h, they decline by 24 h and are at basal levels by 48 h. The increase occurs in the absence of a measurable effect of TSH on DNA synthesis related to cell growth, since [3H] thymidine incorporation into DNA is still at basal levels 24 h after TSH challenge and is maximal only at 48 h. A protein(s) whose formation is inhibited by cycloheximide appears to be critical to the ability of TSH to increase malic enzyme mRNA levels. Thus, cycloheximide given 30 min before TSH prevents the hormone-induced increase in malic enzyme mRNA; also, when given 24 h after TSH, cycloheximide accelerates the loss of the TSH-induced increase in malic enzyme mRNA. In neither case does cycloheximide affect beta-actin mRNA levels. A second factor(s) whose formation is prevented by actinomycin-D appears to be important for the decrease in malic enzyme mRNA levels seen 24 and 48 h after TSH challenge. Thus, in experiments in which it is given 24 h after TSH, actinomycin-D preserves the hormone-induced increase in malic enzyme mRNA levels rather than accelerating the decrease, as does cycloheximide. In the same experiment, beta-actin mRNA levels decrease to less than 10-20% of control values over the same period; this factor also, therefore, appears to exhibit some degree of specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation in culture of cells from an avian holocrine secretory gland: preparation of isolated cells and conditions which induce accumulation of malic enzyme

Mammalian sebaceous glands contain cells which are constantly going through a process of cell division, differentiation, and destruction. Birds have an analogous holocrine secretory gland, the uropygial gland, which is an excellent model for mammalian sebaceous glands and for analysis of the regulation of differentiation. Isolated uropygial cells were purified in good yield, and with high viability, after enzymatic digestion of the duck uropygial gland. Almost exclusively progenitor (basal) cells are recovered after separation of isolated cells on a Percoll density gradient; mature uropygial cells are destroyed during preparation of isolated cells. In primary culture, uropygial gland cells grow to confluence and partially duplicate the in vivo differentiation pathway. Malic enzyme activity increases 30-fold during 4 wks in culture, but there is little, if any, accumulation of fatty acid synthase and only a modest deposition of fat droplets. Medium conditioned by chick embryo fibroblasts inhibits the accumulation of malic enzyme without affecting cell growth. The basement membrane components, collagen, laminin, and Matrigel, which stimulate differentiation in other cell systems, were without effect on uropygial gland cultures. Triiodothyronine, cyclic AMP, and dexamethasone together with isobutylmethylxanthine had no effect on cell growth or malic enzyme activity. Epidermal growth factor, which stimulates cell division, increased cell number with no increase in malic enzyme accumulation. Factors which would stimulate further differentiation are missing from our culture system, but may include components of the basal lamina and/or factors secreted by mesenchymal cells.     

Genetic regulation of malic enzyme activity in the mouse

Cytosolic malic enzyme catalyzes the NADP(+)-dependent oxidative decarboxylation of malate to pyruvate and CO2. Additionally, this enzyme produces large amounts of reducing equivalents (NADPH) required for de novo fatty acid synthesis and provides a precursor for oxaloacetate replacement in the mitochondria. Malic enzyme is considered a key lipogenic enzyme and changes in enzyme activity parallel changes in the lipogenic rate. As would be expected, the activity of malic enzyme responds to a variety of dietary and hormonal factors acting mainly on the rate of enzyme synthesis. In the mouse, the structural locus for malic enzyme (Mod-1) is located on chromosome 9. Two alleles reflecting differences in electrophoretic mobility have been identified. This report demonstrates that the amount of hepatic malic enzyme activity is strain-dependent and is regulated by a malic enzyme regulator locus (Mod1r) located on the proximal end of chromosome 12. Two alleles have been identified: Mod1ra, conferring high enzyme activity (C57BL/6J), and Mod1rb, conferring low enzyme activity (C57BL/KsJ). Biochemical studies have demonstrated differences in the apparent Km and Vmax and in specific activity on purification and immunoprecipitation, features that suggest changes in enzyme structure even though no differences were observed by electrophoresis and isoelectric focusing. These combined data suggest that differences in both enzyme quantity and structure may be involved in the genetic regulation of malic enzyme activity in mice.     

Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells

Summary The secretory tissue of the uropygial gland is of the holocrine type, containing both dividing progenitor cells and lipid-filled differentiated cells. In this study, we examined the relationship between cell division and differentiation. The location of dividing cells was determined by autoradiography of tissue sections from ducklings injected intra-abdominally with ³H-thymidine. Only cells on the basal lamina of the tubules contained labeled nuclei. Dividing cells were distributed uniformly over the length of the tubules. Over the next five days, most of the labeled cells migrated to the lumen of the tubules and disappeared. Cells containing the lipogenic enzymes, fatty acid synthase and malic enzyme, were localized either immunocytochemically using affinity-purified antibodies or cytochemically using a specific assay for malic enzyme activity. Fatty acid synthase and malic enzyme were undetectable in dividing basal cells but present at high levels in differentiating and differentiated cells. Thus, basal cells lying along the basal lamina of the tubules were replacing lipid-laden cells that were continually sloughed into the lumens of the tubules. The signals for differentiation and enzyme accumulation appear to be linked to one another and to cessation of cell division.     

Cobalt regulation of heme synthesis and degradation in avian embryo liver cell culture

Inorganic cobalt was found to induce heme oxygenase activity in primary cultures of embryonic chick liver cells and to inhibit the induction of delta-aminolevulinate synthetase by the porphyrinogenic compounds allylisopropylacetamide, dicarbethoxy-1,4-dihydrocollidine, etiocholanolone, phenobarbital, Aroclor (R)1254, and secobarbital. Much smaller concentrations of Co2+ (5 muM) were required to inhibit delta-aminolevulinate synthetase than to induce heme oxygenase activity (50 muM). These effects of Co2+ on heme synthesis and heme degradation were potentiated by depletion of cellular glutathione content as a result of treatment with diethyl maleate. Cobalt inhibition of the induction of delta-aminolevulinate synthetase was of the same magnitude and probably involved the same mechanism as that produced by cobalt heme dimethyl ester and iron heme. The induction of heme oxygenase by cobalt could be blocked by cycloheximide. Plasma protein synthesis was not inhibited in the presence of concentrations of Co2+ which produced inhibition of delta-aminolevulinate synthetase or induction of heme oxygenase. Other metals such as Cd2+ and Cu2+ also inhibited the induction of delta-aminolevulinate synthetase by allylisopropylacetamide. These findings indicate that Co2+ can regulate heme metabolism directly in liver cells without intermediate actions on extrahepatic tissues. It is suggested that regulation of production of delta-aminolevulinate synthetase and heme oxygenase is mediated through the action of the metal ion rather than the metal in the form of a tetrapyrrole chelate.     

Switching in levels of translatable mRNAs for enolase isozymes during development of chicken skeletal muscle

Developmental changes in the levels of translatable mRNAs for alpha and beta enolase subunits in chicken muscle were determined using the rabbit reticulocyte cell-free translation system. The level of translatable alpha mRNA was decreased gradually during the development of the muscle. On the contrary, the levels of translatable beta mRNA was not detected at early embryonic stage, but increased dramatically after hatching. These changes were closely correlated with those of the corresponding enzyme activities. The results suggest that the developmental changes in the activities of alpha alpha and beta beta enolases in chicken muscle arise as the result of changes in the amount of corresponding mRNAs.