Effects of glucose and insulin on HepG2‐C3A cell metabolism

HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2‐C3A cells were cultured under varying levels of glucose (high, low, and glucose‐free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose‐free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose‐free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2‐C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose‐free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose‐free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2‐C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2‐C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347–356. © 2010 Wiley Periodicals, Inc.     

Substitution of glutamine by glutamate enhances production and galactosylation of recombinant IgG in Chinese hamster ovary cells

The effect of ammonia on Chinese hamster ovary (CHO) cell growth and galactosylation of recombinant immunoglobulin (rIgG) was investigated using shaking flasks with serum free media containing 0–15 mM NH₄Cl. The elevated ammonia inhibited cell growth and negatively affected the galactosylation of rIgG. At 15 mM NH₄Cl, the proportions of monogalactosylated glycan with fucosex (monogalactosylated glycan with fucose) and digalactosylated glycan with fucose (G2F) were 23.9% and 6.3% lower than those at 0 mM NH₄Cl, respectively. To reduce ammonia formation by cells, glutamate was examined as a substitute for glutamine. The use of glutamate reduced the accumulation of ammonia and enhanced the production of rIgG while depressing cell growth. At 6 mM glutamate, ammonia level did not exceed 2 mM, which is only one third of that at 6 mM glutamine. Also, a 1.7-fold increase in the titer of rIgG and specific rIgG productivity, q _rIgG, was achieved at 6 mM glutamate. The galactosylation of rIgG was favorable at 6 mM glutamate. The proportion of galactosylated glycans, G1F and G2F, at 6 mM glutamate was 59.8%, but it was 50.4% at 6 mM glutamine. The use of glutamate also increased complement-dependent cytotoxicity activity, one of the effector functions of rIgG. Taken together, substitution of glutamine by glutamate can be considered relevant for the production of rIgG in CHO cells since glutamate not only enhances q _rIgG but also generates a higher galactosylation essential for the effector function of rIgG.     

Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in non-proliferating primary monolayer cultures

Parenchymal cells from normal adult rat liver, prepared with high yield (30 × 10⁶ cells/g liver) and viability index (>96%) by a non-perfusion method, were maintained in non-proliferating monolayer culture. Several metabolic functions were investigated for 7 days to evaluate functional integrity of the cultured hepatocytes. Leucine was linearly incorporated into protein for 4.5 h at each day of cultivation and the incorporation rate increased up to 2-fold after 3 days. Urea production was maintained at a rate of 0.5 μmoles/mg protein × h for at least 7 days, and its amount was enhanced 2-fold within 24 h by the addition of 3 mM NH₄Cl. Glucose was formed during the first days by the hepatocytes and was then taken up with increasing amount from the surrounding medium. Lactate consumption, on the other hand, was replaced by lactate production after one day of cultivation.Variations in enzyme levels of lactate dehydrogenase, arginase, glutamine synthetase and glucose-6-phosphatase were also studied during the whole culture period. Cell leakage, which was detected only in the case of lactate dehydrogenase (LDH), occurred through the 4th day along with a concomitant loss of intracellular LDH activity. After 4 days, however, the enzyme activity returned to the initial level. Arginase was maintained throughout the cultivation period and was stimulated 2- to 3-fold within 24 h by NH₄Cl. Glutamine synthetase declined within the first 4 h of cultivation and then remained in the hepatocytes with a transitory rise after 2 days. Its activity was also found to be inversely related to the concentration of glutamine in the culture medium up to 4 mM. Glucose-6-phosphatase gradually decreased during the cultivation period, the enzyme activity, however, was stimulated by glucagon within 24 h.     

Construction and evaluation of drug-metabolizing cell line for bioartificial liver support system

Focusing on drug metabolism in liver, we constructed and evaluated a drug-metabolizing bioartificial liver (BAL) support system. In a previous study, we constructed ammonia-metabolizing CHO and hepatoma-derived HepG2 cell lines by recombination of the glutamine synthetase (GS) gene. For further mimicking of liver metabolism, the human hepatoma-derived cell line HepG2 was transformed by the pBudCE-GS-CYP3A4 vector, which contains GS and drug-metabolizing CYP 3A4 genes. The constructed GS-3A4-HepG2 cell line showed 3A4 activity higher than that of human primary hepatocytes. The drug-metabolizing activity of BAL (BAL clearance) was evaluated using this cell line. The estimated clearance was higher than that of the human hepatocyte system.     

Spheroid formation and functional restoration of human fetal hepatocytes on poly-amino acid-coated dishes after serial proliferation

Primary human fetal hepatocytes proliferated in monolayer culture up to the 9th passage. During proliferation, the cells changed their morphology from a fibroblast-like shape after inoculation to an epithelia-like polygonal shape after they reached confluence. The proliferation was associated with the loss of ammonia detoxification capacity, which is essential for the function of bioartificial liver. The cells formed spheroids on a poly-glutamic acid- or poly-aspartic acid-coated polystyrene dish that had a negatively charged surface at neutral pH. However, the cells did not form spheroids on a poly-lysine- or poly-arginine-coated dish that had a positively charged surface, which is reportedly suitable to form spheroids for adult hepatocytes. The activity of cytochrome P450 (CYP 1A1, CYP1A2) of the cells in spheroid culture was about twice as high as that of the cells in monolayer culture. The ammonia detoxification activity of the cells was restored in spheroid culture by treatment with 2% dimethylsulfoxide. These results suggest that the conditions for human fetal hepatocytes to form spheroids are different from that for adult hepatocytes, and the use of poly-glutamic acid or poly-aspartic acid coating may improve spheroid culture of proliferative human fetal hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.     

Effects of elevated ammonia concentrations on survival, metabolic rates, and glutamine synthetase activity in the Antarctic pteropod mollusk Clione limacina antarctica

Information on the effects of elevated ammonia on invertebrates in general, and polar Mollusks in particular, is scant. Questions of ammonia sensitivity are interesting for several reasons, particularly since predicted global change scenarios include increasing anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species are often lipid-rich, and authors have speculated that there is a linkage between elevated levels of lipids/trimethylamine oxide and enhanced ammonia tolerance. In the present study, we sought to examine ammonia tolerance and effects of elevated exogenous ammonia on several key aspects of the physiology and biochemistry of the pteropod mollusk, Clione limacina antarctica. We determined that the 96-h LC₅₀ value for this species is 7.465?mM total ammonia (Upper 95% CL?=?8.498?mM and Lower 95% CL?=?6.557?mM) or 0.51?mg/L as unionized ammonia (NH₃) (at a pH of 7.756). While comparative data for mollusks are limited, this value is at the lower end of reported values for other species. When the effects of lower ammonia concentrations (0.07?mM total ammonia) on oxygen consumption and ammonia excretion rates were examined, no effects were noted. However, total ammonia levels as low as 0.1?mM (or 0.007?mg/l NH₃) elevated the activity of the ammonia detoxification enzyme glutamine synthetase by approximately 1.5-fold. The values for LC₅₀ and observable effects on biochemistry for this one species are very close to permissible marine ammonia concentrations, indicating a need to more broadly determine the sensitivity of zooplankton to potential elevated ammonia levels in polar regions.     

Expression and amplification of glutamine synthetase gene endows HepG2 cells with ammonia-metabolizing activity for bioartificial liver support system

To establish the ammonia-metabolizing cell lines for a bioartificial liver support system, CHO-K1 and HepG2 were transformed with pBK-CMV-GS vector that contains glutamine synthetase (gs) gene. The recombinant cell lines were selected under the various concentrations of glutamine synthetase inhibitor, methionine sulfoximine (MSX). The host CHO-K1 and HepG2 cell lines produces ammonia, but the both MSX tolerable CHO (GS-CHO) and HepG2 (GS-HepG2) cell lines endowed with the high GS activity could metabolize the ammonium from medium. The ammonia-metabolizing activity of CHO and HepG2 cell was about one-fourth of that of primary hepatocyte.     

Role of glutamine and arginase in protection against ammonia-induced cell death in gastric epithelial cells

Ammonia is a cytotoxic factor produced during Helicobacter pylori infection that may reduce the survival of surface epithelial cells. Here we examine whether ammonia kills cells and whether L-glutamine (L-Gln) protects against cell death by stimulating ammonia detoxification pathways. Cell viability and vacuolation were quantified in rat gastric epithelial (RGM1) cells incubated with ammonium chloride at pH 7.4 in the presence or absence of L-Gln. Incubation of RGM1 cells with ammonium chloride caused a dose-dependent increase in cell death and vacuolation, which were both inhibited by L-Gln. We show that RGM1 cells metabolize ammonia to urea via arginase, a process that is stimulated by L-Gln and results in reduced ammonia cytotoxicity. L-Gln also inhibits the uptake and facilitates the extrusion of ammonia from cells. Blockade of glutamine synthetase did not reduce the survival of RGM1 cells, demonstrating that the conversion of L-glutamate and ammonia to L-Gln is not involved in ammonia detoxification. Thus our data support a role for L-Gln and arginase in protection against ammonia-induced cell death in gastric epithelial cells.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.     

Ammonia assimilation and oxygen evolution by a reconstituted chloroplast system in the presence of 2-oxoglutarate and glutamate

(Ammonia plus 2-oxoglutarate)-dependent O₂ evolution by intact chloroplasts was enhanced three- to five fold by 2 mM L- and D-malate, attaining rates of 9–15 μmol mg^-1 Chl h^-1. Succinate and fumarate also promoted activity but D-aspartate and, in the presence of aminooxyacetate, L-aspartate inhibited the malate-promoted rate. A reconstituted chloroplast system supported (ammonia plus 2-oxoglutarate)-dependent O₂ evolution at rates of 6-11 μmol mg^-1 Chl h^-1 in the presence of MgCl₂, NADP(H), ADP plus Pi (or ATP), ferredoxin and L-glutamate. The concentrations of L-glutamate and ATP required to support 0.5 V _max were 5 mM and 0.25 mM, respectively. When the reaction was initiated with NH₄Cl, O₂ evolution was preceded by a lag phase before attaining a constant rate. The lag phase was shortened by addition of low concentrations of L-glutamine or by preincubating in the dark in the presence of glutamate, ATP and NH₄Cl. Oxygen evolution was inhibited by 2 mM azaserine and, provided it was added initially, 2 mM methionine sulphoximine. The (ammonia plus 2-oxoglutarate)-dependent O₂ evolution was attributed to the synthesis of glutamine from NH₄Cl and glutamate which reacted with 2-oxoglutarate in a reaction catalysed by ferredoxin-specific glutamate synthase using H₂O as the ultimate electron donor. The lag phase was attributed to the establishment of a steady-state pool of glutamine. L-Malate did not affect the activity of the reconstituted system.     

A novel function of connexin 32: marked enhancement of liver function in a hepatoma cell line

Connexin 32 (Cx32) is the main gap junction protein in hepatocytes and plays an important role in the regulation of signal transfer and growth control in the liver by constructing gap junction channels and gap junctional intercellular communication (GJIC). In this study, the human Cx32 gene was transfected into a hepatoma cell line (HepG2) that showed aberrant expression of Cx32 and was deficient in GJIC. Cx32-transfected HepG2 not only expressed a higher level of Cx32 mRNA, but also showed increased GJIC compared with HepG2 and vector-transfected HepG2. Furthermore, the liver functions of ammonia removal and albumin secretion of HepG2 were markedly enhanced with Cx32 gene transfection. It may be expected to improve the cellular functions of the hepatoma cell line by Cx32 gene transfection and serve to develop an efficacious bioartificial liver.     

Precursors of glutamic acid nitrogen in primary neuronal cultures: Studies with15N

We utilized gas chromatography-mass spectrometry to study the transfer of¹⁵N from [2-¹⁵N]glutamine, [¹⁵N]leucine, [¹⁵N]alanine, or¹⁵NH₄Cl to [¹⁵N]glutamate and [¹⁵N]aspartate in cultured cerebrocortical GABA-ergic neurons from the mouse. Initial rates of¹⁵N appearance (atom % excess) were somewhat higher with 2mM [2-¹⁵N]glutamine as a precursor than with 1mM [¹⁵N]leucine or 1mM [¹⁵N]alanine, but initial net formation (nmol [¹⁵N]glutamate/mg protein.min^–1) was roughly comparable with all precursors. At steady-state¹⁵N labeling was about two times greater with 2mM [2-¹⁵N]glutamine as precursor. The subsequent transfer of¹⁵N from glutamate to aspartate was extremely rapid, the labelling pattern of these two amino acid pools being virtually indistinguishable. We observed little reductive amination of 2-oxo-glutarate to yield [¹⁵N]glutamate in the presence of 0.3mM¹⁵NH₄Cl. Reductive amination through glutamate dehydrogenase was much more prominent at a concentration of 3.0mM¹⁵NH₄Cl. Glutamate formation via reductive amination was unaffected by inclusion of 1 mM 2-oxo-glutarate in the incubation medium. These results indicate that glutamate synthesis in cultured GABA-ergic neurons is derived not only from the glutaminase reaction, but also from transamination reactions in which both leucine and alamine are efficient N donors. Reductive amination of 2-oxo-glutarate in the glutamate dehydrogenase pathway plays a relatively minor role at lower concentrations of extracellular ammonia but becomes quite active at 3mM ammonia.     

Lactate Contributes to Ammonia-Mediated Astroglial Dysfunction During Hyperammonemia

Even though ammonia is considered to underlie nervous system symptoms of dysfunction during hyperammonemia, lactate, which increases as a metabolic consequence of high ammonia levels, might also be a contributing factor. The data presented here show that NH₄Cl (5 mM) mediates astroglial cell swelling, and that treatment with NH₄Cl or lactate (25 mM) causes rearrangements of actin filaments and reduces astroglial glutamate uptake capacity. Co-application with BaCl₂, which blocks astroglial uptake of NH₄ ⁺, prevents NH₄Cl-mediated cell swelling and rearrangement of actin filaments, but does not reduce NH₄Cl-induced glutamate uptake capacity inhibition. Neither NH₄Cl nor lactate affected glutamate uptake or protein expression in microglial cultures, indicating that astroglial cells are more susceptible to the neurotoxic affects of ammonia. Our results suggest that ammonium underlies brain edema, but that lactate can contribute to some of the cellular dysfunctions associated with elevated cerebral levels of ammonia.     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH₄⁺ is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg²⁺. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg²⁺.Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH₄⁺, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH₄⁺ produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.