Glucose feeding and exercise in trained rats: mechanisms for glycogen sparing

This investigation studied the effect of an oral glucose feeding on glycogen sparing during exercise in non-glycogen-depleted and glycogen-depleted endurance-trained rats. The non-glycogen-depleted rats received via a stomach tube 2 ml of a 20% glucose solution labeled with [U-14C]glucose just prior to exercise (1 h at 25 m/min). Another group of rats ran for 40 min at higher intensity to deplete glycogen stores, after which they received the same glucose feeding and continued running for 1 h at 25 m/min. The initial 40-min run depleted glycogen in heart, skeletal muscle, and liver. In the non-glycogen-depleted rats the glucose feeding spared glycogen in the liver, primarily from the oxidation of blood-borne glucose in muscle. In the glycogen-depleted rats, muscle glycogen was repleted after the feeding, but sources other than the administered glucose also contributed to glycogen synthesis. The results suggest that glycogen depletion rather than the glucose feeding per se stimulates glycogen resynthesis in muscle during exercise in endurance-trained rats.     

Artificial capillary perfusion cell culture: Metabolic studies

Summary Glucose, lactic-acid, and oxygen metabolism of BHK and L929 cells on artificial capillary perfusion units have been studied using several different modes of perfusion. After 7 to 10 days, cells planted in the extracapillary compartment of culture units containing 80 to 150 fibers reached populations that used 0.073±0.025 μmol per min glucose and 0.76±0.26 μl per min oxygen and excreted 0.078±0.038 μmol per min lactic acid. From these data it is estimated that these units contain approximately 2×10⁷ cells. The metabolic rate of cultures perfused through the capillaries or through the extracapillary compartment was not affected significantly by change in flow rate except at perfusion flow rates ≤0.05 ml per min. The cell population, as measured by metabolic activity, did not increase significantly when the serum content of the medium was ≤1%. No major differences were found in glucose utilization rates of equal numbers of cells on artificial capillaries, on short-term suspension culture, or as monolayers in plastic flasks. Artificial capillary perfusion may provide a simple system for studying metabolism of mammalian cells in culture. Research was supported by the U.S. Army Medical Research and Development Command, Washington, D.C. 20314, under Contract No. DAMD 17-76-C-5075.     

Trehalose absorption related with trehalase in developing ovaries of the silkworm,Bombyx mori

Summary The mechanism of trehalose absorption was examined in developing ovaries of the silkworm,Bombyx mori. Trehalose and glucose absorption followed saturation kinetics giving an apparentK _m value of 8.4 mM and a V_max of 12.5 moles/30 min per g ovaries for trehalose absorption, and an apparentK _m value of 26.4 mM and a V_max of 36.6 moles/30 min per g ovaries for glucose uptake. Trehalose absorption was clearly inhibited by addition of NaCN or NaN₃ to the incubation medium.Cellobiose, maltose, sucrose and turanose were taken up by ovaries at much lower rates than trehalose. Among the disaccharidases which hydrolyse these sugars, trehalase activity was highest. The correlation between trehalase activity and trehalose absorption rate was also demonstrated by a reduction of trehalase activity accompanied by reduced absorption rates after extirpation of the suboesophageal ganglion (SG). During trehalose absorption, glucose was released into the incubation medium, but after SG removal, no liberation of glucose was observed. Furthermore, no accumulation of¹⁴C-trehalose, added to the medium, was observed in the cells and almost all radioactivity was recovered as glucose and glycogen in the ovaries.These results suggest that in developing silkworm ovaries, trehalose is absorbed by a specific carriermediated and energy-dependent system, in which the hydrolysis by trehalase is an obligatory step.     

Micropropagation of globe artichoke (Cynara scolymus L.) from underground dormant buds (“ovoli”)

Summary Side shoots excised from underground dormant buds ofCynara scolymus L. were used as primary explants to establishin vitro cultures. A 3×3 factorial experiment with all possible combinations of three concentrations (0.5, 1.0, 2.0 mg/liter or 2.22, 4.44, 8.88 μM) ofN ⁶-benzyladenine (BA) and three concentrations (0, 0.1, 0.2 mg/liter or 0, 0.54, 1.07 μM) of 1-naphthaleneacetic acid (NAA) was used to determine the optimum growth regulator combination for shoot multiplication. The highest rate of axillary shoots was induced on Murashige and Skoog agar medium supplemented with 0 mg NAA/liter and 1.0 mg BA/liter (4.44 μM). Other cytokinins tested (kinetin, zeatin, and 2-isopentenyl-adenine were less effective than BA in inducing axillary shoot growth. Up to 60% of elongated microshoots rooted after 5 weeks on 1/2 MS agar medium supplemented with 2 mg/liter (11.42 μM) indole-3-acetic acid (IAA). Seventy percent of rooted plantlets were transferred successfully into soil. Plants are under evaluation for their genetic uniformity and clonal fidelity.     

A comparison of the ability of normal liver,a premalignant liver,a solid hepatoma and the zajdela ascitic hepatoma,to take up amino acidsin vitro

Summary The net total uptake of several amino acids at low (0.8–3.1 moles/liter) as well as high (800–1200 moles/liter) extracellular concentrations, by normal rat liver, a premalignant liver, a solid hepatoma, and the Zajdela ascitic hepatoma cells, has been compared under conditions in which protein synthesis continues. At low amino acid concentrations, the initial (3 min) total uptake of the various amino acids in the Zajdela cells, was 3–10 (average 7) times more, and the intracellular concentration of the labeled amino acids taken up 14–45 (average 31) times more, than in normal liver. At the high amino acid concentrations, the total uptake in the Zajdela cells, at 60–120 min was 2–5 (average 3.5) times more, and the intracellular concentration of the amino acids taken up 8–19 (average 13) times more, than in normal liver; the corresponding values for the premalignant liver and the solid hepatoma were in between those for normal liver and the Zajdela cells. Further, the rate of the total uptake of amino acids, their intracellular concentration, the proportion of the amino acid taken up utilized for protein synthesis, the rate of incorporation of the amino acid taken up into protein, and the cellular growth rate, seemed to be correlated in the four cell/tissue preparations studied. In most cases, the rate of the net uptake fell drastically with time, the uptake virtually stopping after 90–180 min, probably due to lack of serum in the incubation medium.     

The ability of the rumen ciliate protozoan Diploplastron affine to digest and ferment starch

The ciliate Diploplastron affine is known as a common species of the rumen fauna in cattle and sheep. This protozoon is able to digest cellulose, whereas its amylolytic activity is not well known. The objective of the reported studies was to examine the ability of D. affine to digest starch and to use this polysaccharide to cover the requirement for energy. The enzymatic studies showed that the protozoal cell extract degraded starch to reducing products with the rate being equivalent to 2.4 ± 0.47 μmol/L glucose per mg protein per min. Maltose, maltotriose and a small quantity of glucose were the end products of starch degradation. The degradation rate of maltose was only 0.05 μmol/L glucose per mg protein per min. Two peaks in α-amylase and a single peak in maltase activity were found following molecular filtration of ciliate cell extract, whereas three starch-degrading enzymes were identified by a zymographic technique. Incubation of the bacteria-free ciliates with starch in the presence of antibiotics resulted in a release of volatile fatty acids with the net rate of 25 pmol per protozoan per h. Acetic acid followed by butyric acid was the main product of starch fermentation. The results confirmed the ability of D. affine to utilize starch in energy-yielding processes.     

GLYCOGEN METABOLISM AND CHANGES IN THE ACTIVITIES OF PHOSPHORYLASE, PHOSPHOFRUCTOKINASE AND PYRUVATE KINASE DURING DEVELOPMENT OF SEA URCHIN EGGS

The glucose and glycogen contents of sea urchin eggs and embryos were measured enzymatically. Unfertilized eggs of Hemicentrotus pulcherrimus and Anthocidaris crassispina contain about 20.9 and 24.4 μg of glycogen per mg protein, respectively. As for glucose, unfertilized eggs of Hemicentrotus and Anthocidaris contain about 0.7 and 1.9 μg per mg protein, respectively. Glycogen consumption during embryonic development differs with different species of sea urchins. In Anthocidaris , glycogen decreases significantly after fertilization. The oxidation of glucose and glycogen accounts for about 50% of oxygen consumed until the early blastula stage in this species. The contribution ratio of glucose and glycogen to the overall energy pool becomes less than 10% at later stages. In Hemicentrotus , however, the glycogen content remains unchanged until the early blastula stage and thereafter decreases. The importance of glucose and glycogen as an energy fuel seems little throughout the development of Hemicentrotus. Activities of phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) were measured at various embryonic stages in both species of sea urchins. The difference between two species in the consumption of glucose and glycogen can not be elucidated by the differences in the activities of these enzymes.     

SOME PROPERTIES OF ISOLATED NEURONAL CELL FRACTIONS

Abstract— 1. Histochemical evidence was presented illustrative of the composition of neuronal and neuropil ('glial') fractions isolated according to a previously published procedure. The neuropil refers to all cortical tissue other than neuronal perikarya.
2. On the basis of cell counts and of DNA content, an average cell mass of 100-110 pg was calculated for cells in the neuronal fraction. Eight per cent of the total DNA was recovered in the neuronal fraction.
3. Both fractions synthesized ATP in vitro. Concentrations after 60 min incubation with glucose were: neuropil, 7–36 μmoles/mg protein; neuronal, 12–31 μmoles/mg protein.
4. Osmotic shock or homogenization resulted in changes in turbidity of the cell fractions which were interpreted as indicative of loss of cell structure. The free pool amino acids glutamate, glutamine, GABA, aspartate and alanine were retained in the precipitable material through several washes with isotonic solutions. Homogenization released 72 per cent of the neuronal and 68 per cent of the neuropil amino acids into the supernatant, but only 37 per cent and 19 per cent respectively of the protein.
5. By contrast with earlier reports, K⁺ accumulation has now been demonstrated in both neuronal and neuropil fractions. After incubation with glucose, K⁺ level were calculated as being 80 per cent of slice in the neuronal, and 65 per cent in the neuropil fraction. These results, and those of the osmotic shock experiments, were taken as indicative of the retention of some cell structure.
6. By comparison, cell fractions prepared by other procedures, using acetone-glycerol-water or tetraphenylboron for tissue disaggregation, produced preparations with limited metabolic capabilities; oxygen uptake, CO₂ and lactate production were all lowered substantially.     

Ethanol stimulates glycogenolysis in livers from fed rats.

To determine the reason for the lack of a hypoglycemic effect of ethanol in the fed state, the effect of ethanol on glucose turnover, liver glycogenolysis, and glucose metabolites was determined. Chronically catheterized awake and freely moving fed rats received either ethanol (blood ethanol, 37 +/- 10 mmol/liter, n = 11) or saline (n = 13) intravenously for 4 hr. Glucose turnover was determined using a primed continuous infusion of [3-3H]glucose. The liver was freeze clamped at 4 hr for glycogen and metabolite measurements. Plasma glucose (5.8 +/- 0.3 mmol/liter vs 6.3 +/- 0.2 mmol/liter at 4 hr, ethanol versus saline) and the rate of glucose turnover (61 +/- 9 vs 58 +/- 8 moles/kg.min) were similar during the ethanol and saline infusions. Plasma lactate was significantly higher in the ethanol (1.32 +/- 0.05 mmol/liter) than in the saline (0.86 +/- 0.06 mmol/liter, P less than 0.001) study. Concentrations of gluconeogenic intermediates in the liver (glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, and pyruvate) were all significantly and -30% lower in ethanol-infused than in saline-infused rats. The liver citrate content was similar in ethanol-infused than in saline-infused rats. The liver citrate content was similar in ethanol (0.38 +/- 0.03 mmol/liter) and saline (0.37 +/- 0.04 mmol/liter) studies. Liver glycogen was 75% lower in the ethanol-infused (61 +/- 9 mmol/kg dry wt) than the saline (242 +/- 27 mmol/kg dry wt, P less than 0.001)-infused rats. These data demonstrate that in fed rats given ethanol, glucose turnover is maintained constant by accelerated glycogenolysis. Thus, inhibition of gluconeogenesis by ethanol does not lower hepatic glucose production unless compensatory glycogenolysis can be prevented.     

Effect of surfactants on pyrene degradation by Pseudomonas fluorescens 29L

The effect of surfactants on pyrene degradation in Pseudomonas fluorescens 29L was investigated. This strain produced 30.1 μM of rhamnolipid equivalents (RE) of biosurfactants on 50 mg of pyrene per liter of medium. The production of biosurfactants was significantly correlated with the water solubility (S _w) of the substrate and the growth rate on it. When chrysene, with a S _w of 2.8 × 10⁻³ mg per liter of water, was the carbon source, 13.1 μM of RE of biosurfactants were produced compared to 10.3 μM of RE of biosurfactants on acenaphthene with a S _w of 1.9 mg per liter of water. No biosurfactants were produced on salicylic acid, catechol, and citrate. All of the strain 29L mutants which grew on pyrene produced biosurfactants while among the mutants which grew on naphthalene, only 88.4% produced biosurfactants. The rhamnolipid mixture, JBR425, inhibited the growth of Strain 29L wild type (WT) and all of its mutants on pyrene. However, these mutants were able to grow in the presence of pyrene when the growth medium was supplemented with 10⁻⁶ mg of emulsan per milliliter of medium. This study implies biosurfactants are produced by Strain 29L as a physiological response to the hydrophobicity of pyrene. The combined use of indigenous biosurfactants and the added biosurfactant, emulsan, is a biotechnology to enhance pyrene degradation by Pseudomonas fluorescens 29L.     

Effects of pregnenolone,cyanoketone, and equilenin on the human malignant trophoblast in vitro

Summary Cultures of human malignant trophoblast cells were studied to determine the basis of inhibition of human chorionic gonadotropin (HCG) secretion and depletion of glycogen following incubation of the cells in the presence of pregnenolone (3β-hydroxypregn-5-en-20-one). Incubation of the cells for as long as 3 days with 5 or 10 μg of pregnenolone per ml resulted in decreased protein content, inhibition of DNA synthesis, diminished glucose utilization, and marked accumulation of acellular debris in the medium. These changes became more pronounced with time of incubation and were related to the dose of pregnenolone employed. The effect of pregnenolone on all of the parameters measured was mimicked and potentiated by either equilenin (3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one) or cyanoketone (androst-5-en-2α-cyano-17β-hydroxy-4,4,17α-trimethyl-3-one), inhibitors of pregnenolone conversion to progesterone. These results suggested that the glycogenolysis and inhibition of HCG secretion that occur when the trophblast cells are incubated in the presence of pregnenolone result from toxicity rather than from cellular differentiation, and that prior conversion of pregnenolone to progesterone is not necessary for the manifestation of the toxicity. This work was supported in part by Contract PH 43-NCI-E-68-1010 from the Special Virus Cancer Program, National Cancer Institute, United States Public Health Service.     

Tunicamycin inhibition of <Emphasis Type="Italic">N</Emphasis>-glycosylation of α-glucosidase from <Emphasis Type="Italic">Aspergillus niveus</Emphasis>: partial influence on biochemical properties

Treatment of Aspergillus niveus with 30 μg tunicamycin/ml did not interfere with α-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme had similar molecular masses, ~56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65°C). The K_m and V_max values of under- and fully-glycosylated forms of α-glucosidase were similar when assessed for hydrolysis of starch (~0.6 mg/ml, ~350 μmol glucose per min per ml), maltose (~0.54 μmol, ~330 μmol glucose per min per ml) and p-nitrophenyl-α-d-glucopyranoside (~0.54 μmol, ~8.28 μmol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.     

Studies on gluconeogenesis and protein synthesis in isolated hepatocytes.

Glucose production was studied in isolated hepatocytes using various substrates and with increasing substrate concentrations (0-10 mM). Fructose was the best gluconeogenic substrate while other substrates studied stimulated net glucose production in the following decreasing order: lactate, pyruvate, glycerol, galactose, alanine, and succinate. Studies on oxygen consumption showed that endogenous respiration was linear for 60 min and was not altered by extracellular calcium. Studies on the incorporation of 14C-leucine into protein was linear for only 3-4 hr in cells containing low glycogen. However, cells containing high glycogen incorporated 14C-leucine into protein linearly for 8-10 hr. About 3 mg of protein per g per hr was synthesized by isolated cells when incubated for 4 hr with amino acids mixture, glucose, lactate, and insulin.     

Rate and duration of net glycogen synthesis following glucose administration to fasted human leukocytes

When glucose was added to fasted human leukocytes in a final concentration of 0.5-5 mM there was a phase of glycogen synthesis followed by a phase of glycogen breakdown. The duration of the phase of net glycogen synthesis increased with increasing concentrations of glucose applied, but the net rate of glycogen synthesis was inversely related to this figure and decreased from approx. 7 nmol/10(7) cells per min at 0.5 mM glucose to an average of 4 nmol/10(7) cells per min at 5 mM glucose.     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement of P450(BM-3) whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli

Escherichia coli BL21, expressing a quintuple mutant of P450_BM-3, oxyfunctionalizes α-pinene in an NADPH-dependent reaction to α-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP⁺-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial α-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g⁻¹ cell dry weight after 1.5 h. The initial total product formation rate was 1,000 μmol h⁻¹ μmol⁻¹ P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased α-pinene oxide yield in the presence of glucose compared to glucose-free conditions.     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations.

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations.

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.     

Cytophotometric analysis of glycogen,protein and DNA of a glycogen-storing rat hepatoma (N13) cell line

This study examines the behavior of glycogenstoring rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G₂/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N⁶,O²-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G₀/G₁ and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.