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.     

A biochemical and stereological study of neonatal rat hepatocyte subpopulations. Effect of pre- and postnatal exposure to ethanol

Hepatocytes from 12-day-old rats, pre- and post-natally exposed to alcohol, together with those from pair-fed controls, were isolated and subfractionated in six cell subpopulations on Percoll density gradients. These cells were characterized using a combination of biochemical and stereological methods. The low density cells (F2) mainly showed biochemical and stereological features of perivenous hepatocytes, whereas the heavier cells (F6) were primarily periportal hepatocytes. The alcohol-metabolizing enzymes, alcohol dehydrogenase and aldehyde dehydrogenase (high and low Km) showed more activity in the F2 fraction. Alcohol-altered mitochondria and Golgi apparatus occurred mainly in F2 cells, whereas the endoplasmic reticulum and lysosomes appeared to be more altered in the F6 hepatocytes. Alcohol also induced the appearance of some small hepatocytes, with a well-developed rough endoplasmic reticulum and an increased number of mitochondria. Biochemical data indicated that glutamate dehydrogenase and alanine aminotransferase were more affected in F2 cells from alcohol-treated rats, and that the activity of the ethanol-metabolizing enzymes was alos reduced in these hepatocytes. Our results indicate that alcohol exposure during zonal development in the liver could have a selective effect on specific cell components depending on the acinar zone, and that the perivenous hepatocytes appear to be more damaged under these conditions.     

Maintenance of glucokinase activity in primary hepatocyte cultures

When primary cultures of hepatocytes are maintained for 2 weeks from the time of perfusion, the activity of the enzyme glucokinase decreases rapidly, so that the activity can no longer be detected after the fourth day in culture. Concomitantly, there occurs an increase in the activity of hexokinases, the low-K_M isozymes, which predominate in fetal liver. We have made several modifications of the culture medium in an attempt to prevent the decrease in glucokinase activity. When the medium was supplemented with a mixture of insulin, thyroxine, glucagon, dexamethasone, testosterone, and estradiol, the activity of the enzyme in the hepatocytes was present at approximately 15% of in vivo levels after 2 weeks in culture. When this hormone mixture was present during the first 4 hrs of culture and when the hepatocytes were allowed to attach to the collagen support and were maintained thereafter in medium supplemented with fetal bovine serum, insulin, and dexamethasone, the activity of glucokinase increased after an initial decrease for 3 days and was maintained thereafter at levels comparable to those observed in vivo. This effect of the hormone mixture was found to be the result of the presence of glucagon in the mixture, since the presence of glucagon with no other hormones added, except insulin, during the attachment period produced the same pattern of increased glucokinase activity. Immunoprecipitation of glucokinase from the hepatocytes, using monospecific antibody, indicated that the increase in enzyme activity was the result of increased glucokinase enzyme protein and not an increased synthesis of the other hexokinase isozymes. These studies demonstrate the specific hormonal requirements for the maintenance of glucokinase levels in primary hepatocyte culture at those seen in vivo and lends support to the hypothesis that fetal gene expression in primary hepatocyte cultures is selectively regulated rather than being a general effect with a common regulatory mechanism.     

Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway.

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.     

Fasting modulates metabolic responses to cortisol, GH and IGF‐I in Arctic charr hepatocytes

Hepatocytes in primary culture from fed and 2 month fasted Arctic charr Salvelinus alpinus were exposed to physiological doses of either cortisol, salmon growth hormone (GH), salmon insulin‐like growth factor‐I (IGF‐I) or a combination of salmon GH and salmon IGF‐I. Fasting significantly lowered medium glucose levels compared to the fed fish, but had no significant effects on hepatocyte glycogen content or on the activities of enzymes involved in the intermediary metabolism. Cortisol treatment had no effect on hepatocyte glycogen content or on the enzyme activities investigated, but resulted in a significant increase in medium glucose concentration in hepatocytes isolated from fasted, but not fed fish. GH and IGF‐I treatments, both singly and in combination, significantly increased the glycogen content of hepatocytes isolated from fed fish, with less pronounced effects on hepatocytes isolated from fasted fish. The combination of GH and IGF‐I significantly increased lactate dehydrogenase activity regardless of the feeding state and significantly reduced the phosphenolpyruvate carboxykinase activity and medium glucose concentration in hepatocytes isolated from fed fish. Further, GH and IGF‐I significantly increased the activities of alanine aminotransferase and aspartate aminotransferase in hepatocytes isolated from fasted fish, but not fed fish. There were no effects of GH, IGF‐I, or their combination, on glucose 6‐phosphate dehydrogenase or 3‐hydroxyacyl‐CoA dehydrogenase activities. The results demonstrated that nutritional status of the animal modulates hepatocyte responsiveness to metabolic hormones, and suggested a role for GH and IGF‐I in hepatic glycogen conservation.     

Hormonal effects on the biosynthesis of lactate dehydrogenase in rat hepatocytes.

Rat hepatocytes have been studied in suspension culture for 10-h periods. Levels of extractable lactate dehydrogenase (LDH) have been measured in these hepatocytes at hourly intervals in order to note the balance between biosynthesis and degradation of this enzyme. Newly synthesized LDH has been measured by following the rate of incorporation of [3H]leucine into radiochemically pure LDH of high specific catalytic activity as isolated by a rapid affinity chromatographic procedure. The effects of the addition of physiological concentrations of the following hormones at the beginning of 10-h culture periods immediately following preparation of the hepatocytes by the collagen perfusion procedure have been recorded. The hormones triiodothyronine (T3), insulin, glucagon, and dexamethasone have been added singly or in combination. The culture medium has supplied variable amounts of these hormones in the 10% of fetal calf (or other) serum added, and the hepatocytes themselves have provided intracellular amounts of hormones. In addition to the added hormones, N6,O2'-dibutyryl cyclic AMP (Bt2cAMP) has also been studied. Control suspensions of hepatocytes show reproducible initial levels of extractable LDH which are maintained or slightly increased during 10 h. Such control systems also incorporate [3H]leucine into total protein and into highly purified LDH at reproducible rates during 10 h of incubation. The effects of added hormones on LDH lavels are as follows: (a) T3 causes about a 2-fold increase in LDH at 7 to 8 h in hepatocytes from young adult animals, an effect which is lowered in either younger or older animals or in thyroidectomized animals. (b) Insulin leads to a similar increase in LDH at 5 to 6 h and a falling off at 8 to 10 h. (c) Glucagon also causes an approximate doubling of the amount of extractable LDH during a 10-doubling of the amount of extractable LDH during a 10-h period. (d) Dexamethasone does not produce an increase. (e) Bt2-cAMP produces an effect indistinguishable from that of glucagon. Paired combinations of these hormones fail to produce an additive response in any case. The combinations of T3 plus dexamethaseon and insulin plus dexamethasone lead to significant reductions in levels of extractable LDH when compared to the single hormone effects cited above. With respect to rates of synthesis of total protein as measured by [3H]leucine incorporation, only glucagon, glucagon plus Bt2-cAMP, glucagon plus insulin, T3 plus Bt2cAMP, and T3 plus insulin produce significant increases during a 10-h period. However, when [3H]leucine incorporation into highly purified LDH is measured as an index of LDH biosynthesis, T3, insulin, and glucagon consistently increase the biosynthetic rates during a 10-h period. Bt2cAMP produces a smaller increase. Dexamethasone fails to produce any significant change when compared to controls. Paired combinations of hormones again do not produce any additive effect on LDH biosynthesis when the hormone producing the higher level is taken as the reference...     

Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.     

Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.     

Regulation of alcohol oxidase synthesis in Hansenula polymorpha: Oversynthesis during growth on mixed substrates and induction by methanol

The regulation of the synthesis of alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase was investigated in the methanol-utilizing yeast Hansenula polymorpha. The organism was found to synthesize immunologically identical alcohol oxidases during growth on glycerol and methanol. Growth on glycerol, however, was not dependent on the alcohol oxidase, as was shown with a mutant without alcohol oxidase protein. Similarly it was shown with a catalase activity negative mutant that high catalase activity during growth on glycerol was not a prerequisite for the utilization of this substrate, though absolutely required for growth on methanol.Experiments were conducted with mixed substrates to study the influence of methanol on alcohol oxidase synthesis. In batch cultures, growth on ribose plus methanol resulted in an enhanced rate of alcohol oxidase synthesis as compared to ribose alone. In continuous cultures, (D=0.1 h^-1) addition of methanol to glycerol-, glucose-, or sorbose-limited cultures gave rise to increased alcohol oxidase activity of up to 20 U/mg, which is about by 2 times higher than the specific activity used for growth on methanol alone. The increase in specific activity of the dissimilatory enzymes on the mixed substrates is partly due to methanol per se, as was shown by a mutant unable to dissimilate or assimilate methanol.     

Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc.

Zymomonas mobilis is an unusual microorganism which utilizes both iron-containing alcohol dehydrogenase (ADHII) and zinc-containing alcohol dehydrogenase (ADHI) isoenzymes during fermentative growth. This organism is obligately ethanologenic, and alcohol dehydrogenase activity is essential. The activities of ADHI and ADHII were altered by supplementing growth medium with iron or zinc salts and by iron starvation. Growth under iron-limiting conditions (chelators, minimal medium) reduced ADHII activity but did not prevent the synthesis of the ADHII protein. The inactive form of this enzyme appeared quite stable, was not renatured by iron addition, and persisted in the cell. The iron-induced increase in ADHII activity required de novo synthesis which was blocked by antibiotic additions. The ability of Z. mobilis to synthesize ADHII and ADHI may be advantageous in nature.     

Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.     

The mechanism of the hyperglycaemic action of synthetic peptides related to the C-terminal sequence of human growth hormone

Synthetic part sequences of human pituitary growth hormone (hGH 176–191 and hGH 177–191) corresponding to residues 176–191 or 177–191 of the hormone have been tested for their effects on glycogen and pyruvate metabolism in the rat, both in vivo and in vitro. When injected, the peptides caused transient increases in blood glucose and lactate, while decreasing the activity ratio of glycogen synthase in muscle, adipose tissue and liver and of pyruvate dehydrogenase in muscle and adipose tissue, but not in liver. These decreases were associated with the conversion of the enzymes from their active to their inactive forms, since the peptides did not affect the total amount of either the synthase or the dehydrogenase. The time course of the effect on the enzymes was similar to that for the effect on blood metabolites, and responses for synthase were produced over the range 0.07–7 nmols hGH 177–191/kg body weight. Phosphorylase activity was not affected by the peptides, nor was the capacity to dispose of injected L-lactate. Experiments with adipocytes and hepatocytes showed that the peptides also affected glycogen synthase and pyruvate dehydrogenase activities in vitro. The peptides had no effect on the overall rate of gluconeogenesis from lactate by hepatocytes. However, at times corresponding to those at which glycogen synthase was inactivated, the peptides caused increased incorporation of lactate into free glucose and decreased incorporation into glycogen. It was concluded that the peptides acted directly on their target tissues, and that the observed hyperlactataemia was the result of the inactivation of pyruvate dehydrogenase. The addition lactate increased the flux through the gluconeogenic pathway, and appeared as glucose because the peptide also inactivated glycogen synthase. Thus, the hyperglycaemia produced by hGH 177–199 and related peptides is explicable in terms of a modified Cori Cycle.     

The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.