Butyrate-induced glycolipid biosynthesis in HeLa cells: properties of the induced sialyltransferase.

CMP-sialic acid:lactosylceramide sialyltransferase is induced in HeLa cells by butyrate which also causes the cells to undergo morphological changes including the extension of neurite-like processes. The activity of this enzyme is more than 20-fold higher in butyrate-treated cells than in cells grown without this short chain fatty acid. In vitro synthesis of hematoside from endogenous acceptors is also elevated in cells grown in the presence of butyrate. The levels of induced enzyme activity are influenced by the pH of the culture medium, being higher in more acidic cultures, but are not affected markedly by varying the cell density over a wide range. Detergent is required for in vitro sialyltransferase activity, and this activity is stimulated almost fivefold by cardiolipid. The optimum pH for in vitro activity is 6.0 and the apparent K_m value for lactosylceramide is 3.5 × 10^?5m. Although there are several sialyltransferase activities in HeLa cells, the induced enzyme is specific for lactosylceramide.     

Effect of butyrate on glycolipid metabolism of two cell types of rat ascites hepatomas with different ganglioside biosynthesis

The effect of butyrate on glycolipid metabolism and morphological differentiation in the cell culture system of rat ascites hepatomas, AH 7974 of island-forming type and AH 7974F of free type, was studied. Both cell lines adhered to the substratum in the presence of 1 mM butyrate. In the case of AH 7974, the addition of butyrate induced a distinct morphological change but the other cell line showed no such conspicuous change. Butyrate-treated AH 7974 cells showed a 2 to 3-fold elevation of CMP-N-acetylneuraminic acid: lactosylceramide sialyltransferase activity to form N-acetylneuraminylgalactosylglucosylceramide (GM3). On the other hand, no enzyme activity could be detected in AH 7974F cells. Four glycosyltransferase activities involved in glycolipid synthesis, including sialyltransferase in AH 7974F cells, were reduced by butyrate. From these observations we concluded that sialyltransferase to form GM3, or TM3 itself, is prerequisite for the morphological alteration induced by butyrate.     

Increased alkaline phophatase activity in HeLa cells mediated by aliphatic monocarboxylates and inhibitors of DNA synthesis

Alkaline phosphatese activity of HeLa cells is increased from 3- to 8-fold during growth in medium with certain aliphatic monocarboxylates. The four-carbon fatty acid salt, sodium butyrate, is the most effective “inducer” with propionate (C₃), pentanoate (C₅) and hexanoate (C₆) having lesser effects. Other straight-chain aliphatic monocarboxylates, branched-chain analogues of inducers, hydroxylated derivatives, and metabolytes structurally related to butyrate are ineffective in mediating an increase in enzyme activity, indicating stringent structural requirements for inducers. The kinetics of increase in alkaline phosphatase activity in HeLa cells shows a 20–30 h lag period after adding the aliphatic acid followed by a rapid linear increase of enzyme activity. Protein synthesis is required for “induction”. The isozyme of HeLa alkaline phosphatase induced by monocarboxylates is the carcinoplacental form of the enzyme as determined by stereospecific inhibition by the l-enantiomorphs of phenylalanine and tryptophan, heat stability, and immunoreactivity with antibody against the human placental enzyme.Monocarboxylates that mediate increased alkaline phosphatase activity inhibit HeLa cell multiplication. Inhibition of HeLa cell growth may be necessary for induction and this hypothesis is supported by the findings that three different inhibitors of DNA synthesis, i.e. hydroxyurea, 1-β-d-arabinfuranosyl cytosine and methotrexate, also increase alkaline phosphatase activity. These inhibitors are synergistic with butyrate in causing HeLa cells to assume a more spindle-like shape and in producing an up-to 25-fold increase of enzyme activity. Studies on the modulation of carcinoplacental alkaline phosphatase by monocarboxylates commonly used as antimicrobial food additives and by anti-neoplastic agents may provide methods to evoke “tumor markers” of human occult malignancies. These drug-induced elevations of fetal isozyme activity may further our understanding of gene expression in human cells.     

Sodium butyrate induced alterations in lysosomal enzyme activity

Summary Treatment of cultured HeLa cells with 5 mM sodium butyrate causes an inhibition of growth as well as extensive chemical and morphological differentiation. Lysosomal enzyme activity changes have been associated with both normal and neoplastic growth as well as many aspects of the neoplastic process. The comparative ultrastructural results show that the butyrate-treated cells have a more extensive internal membraneous system than the untreated cells, whereas other organelles seem unaffected by the butyrate treatment. Methods for the histochemical localization of lysosomal acid phosphatase show a twofold increase in particulate reaction product in the butyrate-treated HeLa cells. Isolation of lysosomes followed by a comparative enzyme analysis shows a two to three fold increase in acid phosphatase activity per cell after 24 h of butyrate treatment, as well as three to four fold increase in β-glucuronidase activity. These increases reverse within 24 h of removal of the butyrate from the culture medium. These results as interpreted suggest that butyrate treatment may be preventing sublethal autolysis by arresting the leakage of the lysosomal enzymes from the lysosome into the cytosol and thus allowing the cell to chemically and morphologically differentiate. This work was supported by National Institute of Health Grant HD 14085-03.     

Morphological changes in cultured mammalian cells: prevention by the calcium ionophore A23187.

The morphological changes induced by butyrate in HeLa cells and by monobutyryl or dibutyryl cAMP in CHO cells are prevented by micromolar concentrations of the divalent cation ionophore A23187. The ionophore is unable to prevent such changes in medium from which calcium is omitted. At slightly higher (but nontoxic) concentrations, the ionophore inhibits the butyrate-mediated induction of the ganglioside biosynthetic enzyme, sialyltransferase, in HeLa. In CHO, sialyltransferase activity is normally high and not altered by any of the compounds tested.     

Modulation of lipid-synthesizing enzymes by insulin in differentiated ob17 adipose-like cells.

ob17 cells convert into adipose-like cells when maintained in the presence of physiological concentrations of insulin and tri-iodothyronine. After this conversion, insulin removal from differentiated ob17 cells gives within 24-48 h a large decrease in fatty acid synthetase, glycerol 3-phosphate dehydrogenase and acid:CoA ligase activities, as well as in the rate of fatty acid synthesis determined by [14C]acetate incorporation into lipids. All parameters are restored by insulin addition to initial values within 24-48 h. Dose-response curves of insulin on the restoration of glycerol 3-phosphate dehydrogenase activity and of fatty acid synthesis give half-maximally effective concentrations close to 1 nM, in agreement with the affinity for insulin of the insulin receptors previously characterized in these cells. Immunotitration experiments indicate that the changes in the specific activity of fatty acid synthetase are due to parallel changes in the cellular enzyme content. Therefore the ob17 cell line should be a useful model to study the long-term effects of insulin on the modulation of lipid synthesis in adipose cells.     

Morphological and biochemical differentiation in HeLa cells. Effects of cycloheximide on butyrate-induced process formation and ganglioside metabolism.

When butyrate-treated HeLa cells are trypsinized and replated in the absence of butyrate, their neurite-like processes re-extend transiently. Process formation after replating is prevented when the cells are exposed to cycloheximide during butyrate treatment, whereas it is not prevented by prior exposure to the calcium ionophore A23187 plus butyrate. These results indicate that butyrate induces protein(s) required for process extension which can accumulate in the absence of processing and promote processing in the absence of inducer. Transient process re-extension is followed by spontaneous retraction of processes and reversion to normal morphology. Reversion is not prevented or delayed by puromycin. Surprisingly, however, cycloheximide completely prevents reversion even at low concentrations (< 0.5 μg/ml). Levels of the ganglioside sialolactosylceramide (G_M3), synthesis of which is induced by butyrate, return to basal levels after removal of the inducer. Cycloheximide at 0.5 μg/ml prevents the decline of G_M3 levels after removal of butyrate although the biosynthetic enzyme sialyltransferase decays at the same rate in the presence or absence of the drug and the activity of the sialidase is not affected. The results further support the hypothesis that the ganglioside G_M3 is necessary for the morphological differentiation induced in HeLa cells by butyrate.     

Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20-24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-alpha 1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34-48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of alpha-amanitin in the culture media at 0 h. The inhibiting effect of alpha-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.     

Lactosylceramide molecular species specificity of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase

Six naturally occurring and three synthetic molecular species of lactosylceramide (LacCer) were used to examine the molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase in a Golgi-rich fraction of rat liver. The enzyme molecular species specificity was determined either in the presence of nonspecific lipid transfer protein or in the presence of detergents. Assays performed in the presence of transfer protein showed that for those lactosylceramide molecular species with either d18:1 or d18:0 long chain base the enzyme activity decreased linearly as the effective carbon number of the fatty acid increased. An increase in the carbon number of the long chain base decreased the activity of the enzyme twice as much as a corresponding increase in the carbon number of the fatty acid. On the other hand, when the enzyme activity was assayed in the presence of detergents, there was no significant difference in activity among the various molecular species of lactosylceramide based upon the carbon number of the fatty acid or on the presence of a double bond in the long chain base. However, the decrease in enzyme activity with an increase in the carbon number of the long chain base persisted. These results demonstrate that sialyltransferase has binding specificity with respect to the long chain base, but not the fatty acid. The apparent molecular species towards the fatty acid is related to the aqueous solubility of the various LacCer molecular species.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.     

In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235. Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides

The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.     

Temperature dependence of membranous and solubilized sialyltransferase activities in the presence of 1-palmitoyl-sn-glycero-3-phosphorylcholine and fatty acids

The temperature dependence of sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetyl-neuraminyltrasferase, EC 2.4.99.1) inhibition is described when 1-palmitoyl-sn-glycero-3-phosphorylcholine, or a saturated fatty acid (lauric, myristic or palmitic acid) or an equimolar mixture of the two components are added. Lysophospholipid and fatty acids have no appreciable effect on the optimal temperature for sialyltransferase activity. In the presence of lysophospholipid, the membranous sialyltransferase activity is decreased for all the temperature range tested. In contrast, the solubilized sialyltransferase activity is decreased for temperatures exceeding 29 degrees C. In the presence of saturated fatty acids, the membranous activity is decreased above a chain-length dependent temperature: 22 degrees, 25 degrees and 30 degrees C for lauric, myristic and palmitic acids, respectively. In contrast, the solubilized activity remains unchanged. In the presence of equimolar mixtures of lysophospholipid and fatty acid, the membranous activity is decreased above the same critical temperature as that described for fatty acids added alone. In contrast, the solubilized activity is decreased above 29 degrees C. From these observations, it is suggested that lysophospholipid inhibits the solubilized enzyme when the temperature exceeds the critical micellar temperature of this lipid. The fatty acids inhibit the microsomal enzyme probably by incorporating into the membrane. It is also suggested that equimolar mixtures of lysophospholipid and fatty acid give rise to molecular analogs of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine.     

Effect of unsaturated fatty acids on the biosynthesis of glucose-repressed enzymes in yeast

In anaerobically glucose-grown yeast isocitrate lyase (EC 4.1.3.1.), malate synthase (EC 4.1.3.2.) and malate dehydrogenase (EC 1.1.1.37.) are repressed by glucose. 24 h cultures still contain 0.3–0.4% glucose in the medium, which is enough to completely repress these activities. Aeration of these cells, in buffer containing acetate, initiates the formation of the three enzymes. Within 16 h, the specific activities of these enzymes increase about 140, 120 and 70-fold, respectively. Glucose-6-phosphate dehydrogenase activity was not altered. When the yeast was grown anaerobically, but with a supplement of an unsaturated fatty acid in the medium, synthesis of the three enzymes was much faster and the specific activities after 16 h of derepression were considerably higher. A relationship exists between the number of double bonds in the unsaturated fatty acid molecule and its capability to stimulate enzyme synthesis: linolenic acid is more effective than linoleic acid, which, in turn, is much more effective than oleic acid. Increasing periods of aeration with glucose of anaerobically grown cells prior to derepression results in an increasing stimulation of enzyme synthesis on subsequent derepression. Anaerobic incubation of yeast in the presence of an unsaturated fatty acid in advance to derepression also increased the velocity of enzyme formation. It is suggested that during the aeration period with glucose and during anaerobic incubation with an unsaturated fatty acid a more active protein synthesizing apparatus was formed.     

Adaptive Formation of Fatty Acid Activating Enzyme in Chilomonas paramecium1

SYNOPSIS. Chilomonas paramecium contains 2 different fatty acid activating enzymes (FAAE), one of which utilizes acetate as a substrate, while the other catalyzes the reaction with either butyrate or hexanoate. The site of greatest activity of these enzymes was found to be not in the mitochondrion, but in the “soluble” portion of the cell. Synthesis of acetyl FAAE is constitutive; this enzyme is present regardless of the substrate in the growth medium. The synthesis of the butyryl-hexanoyl FAAE is induced by the presence of either of the substrates. The details of induction of the butyryl enzyme in acetate-grown cells, and the de-adaptation of cells grown in butyrate and transferred to acetate, are given. One mole of pyrophosphate is produced for each mole of CoA-SH reacting, (thus establishing the prevalence of the acyl-adenylate pathway in Chilomonas fatty acid activation.     

Serum factors that stimulate fatty acid oxidation: physiological specificity

In the accompanying paper (Wice et al., 1986) we reported that serum from chickens contains small molecular weight compounds that stimulate long-chain fatty acid oxidation ten fold or more in HeLa cells. Here we show that this response is not limited to specific sera or to specific target cells. The specificity of the metabolic response to these factors was also investigated. They had no effect on the following major pathways of HeLa cell metabolism: 1) the oxidation of the medium-chain fatty acid, octanoic acid, 2) the rate of glycolysis of glucose, 3) the flux of glucose carbon through the oxidative arm of the pentose cycle, 4) the entry of pyruvate into the citrate cycle, 5) the oxidation of glutamine carbon, 6) the utilization rate of oxygen or 7) the rate of fatty acid synthesis. Furthermore, the increased oxidation of long-chain fatty acids was not a result of an increased uptake into the cells. Thus, the serum factors appear to be very specific for the oxidation of long-chain fatty acids for energy. Since carnitine also stimulates long-chain fatty acid oxidation in these cells, it seems likely that these compounds either facilitate the activity of carnitine or provide the same function--presumably the transport of long-chain fatty acid into and out of the mitochondria.     

Biosynthesis and intracellular transport of alpha-2,6-sialyltransferase in rat hepatoma cells.

We investigated biosynthesis, intracellular transport and release of beta-galactoside alpha-2,6-sialyltransferase in a dexamethasone-inducible rat hepatoma cell line. Confluent cells were induced by 10 microM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S-labelled enzyme was synthesized as a 46-kDa precursor, converted to an intermediate 47-kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N-glycosylation or cleavage of N-glycans from isolated sialyltransferase using N-glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4-h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N-acetyllactosamine-type or one oligomannose-type and one N-acetyllactosamine-type N-linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4-h chase. The half-life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion-blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments. Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post-Golgi degradation pathway. This route contrasts with the post-Golgi trafficking of beta-1,4-galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623-7628].     

Dexamethasone regulates bile acid synthesis in monolayer cultures of rat hepatocytes by induction of cholesterol 7 alpha-hydroxylase.

To study the effect of steroid hormones on bile acid synthesis by cultured rat hepatocytes, cells were incubated with various amounts of these compounds during 72 h and conversion of [4-14C]cholesterol into bile acids was measured. Bile acid synthesis was stimulated in a dose-dependent way by glucocorticoids, but not by sex steroid hormones, pregnenolone or the mineralocorticoid aldosterone in concentrations up to 10 microM. Dexamethasone proved to be the most efficacious inducer, giving 3-fold and 7-fold increases in bile acid synthesis during the second and third 24 h incubation periods respectively, at a concentration of 50 nM. Mass production of bile acids as measured by g.l.c. during the second day of culture (28-52 h) was 2.2-fold enhanced by 1 microM-dexamethasone. No change in the ratio of bile acids produced was observed during this period in the presence of dexamethasone. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of the bile acid pathway, to bile acids was not affected by dexamethasone. Measurement of cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes, incubated with 1 microM-dexamethasone, showed 10-fold and 90-fold increases after 48 and 72 h respectively, as compared with control cells. As with bile acid synthesis from [14C]cholesterol, no change in enzyme activity was found in hepatocytes cultured in the presence of 10 microM steroid hormones other than glucocorticoids. Addition of inhibitors of protein and mRNA synthesis lowered bile acid production and cholesterol 7 alpha-hydroxylase activity and prevented the rise of both parameters with dexamethasone, suggesting regulation at the mRNA level. We conclude that glucocorticoids regulate bile acid synthesis in rat hepatocytes by induction of enzyme activity of cholesterol 7 alpha-hydroxylase.     

Choline-phosphate cytidyltransferase activity and phosphatidylcholine synthesis in rat granular pneumocytes are increased with exogenous fatty acids

We investigated the effect of exogenous fatty acids on phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) synthesis by rat granular pneumocytes in primary culture. Synthesis of PC and DSPC from [3H-methyl]choline, as evaluated by increasing specific activity (pmol choline incorporated/microgram phosphorus), was linear for 3 h. Exogenous palmitic, oleic, linoleic, or linolenic acid (100 microM each) increased the synthesis of PC by approx. 50% during incubation for 3 h. In contrast, synthesis of DSPC was increased only by palmitic acid. The increase in DSPC synthesis was approx. 150% after 3 h. Conversion of choline phosphate to PC was increased in the presence of palmitic or oleic acid as indicated by pulse-chase studies with [3H-methyl]choline in the intact cells. Cells incubated for 3 h with either oleic or palmitic acid showed increased choline-phosphate cytidyltransferase activity in the cells and the microsomal fraction. In addition, oleic acid increased the activity of this enzyme in the cytosolic fraction. The distribution of this enzyme in cytosolic and microsomal fraction was 24 and 76% in the cells incubated with palmitic acid and 32 and 68% in control cells. These results suggest that exogenous fatty acids stimulate the de novo pathway of PC synthesis in granular pneumocytes by increasing the microsomal choline-phosphate cytidyltransferase activity.