Activities of enzymes of lipid metabolism in Morris hepatoma 7800 C1 cells

(1) The rate of palmitate oxidation in the 7800 C1 Morris hepatoma cells was about 60% of the activity observed in hepatocytes. The stimulatory effect of glucagon in hepatocytes was not observed in the hepatoma cells. The rate of fatty acid synthesis from [2-14C]acetate in the hepatoma cells was 1/20 of the activity in hepatocytes. The conversion of [2-14C]acetate to cholesterol was not different in the two kinds of cell. (2) Acetyl-CoA carboxylase and fatty acid synthetase were significantly decreased in the hepatoma cells. The hepatoma cells had, however, raised activities of malate dehydrogenase (decarboxylating), and glucose-6-phosphate and 6-phosphogluconate dehydrogenases. (3) The activities of the enzymes were not affected by different concentrations of glucose or palmitate in the culture medium. Insulin, dexamethasone, triiothyronine and glucagon had no effect on the enzyme activities. This is in contrast to the adaptation of the peroxisomal beta-oxidation system, which is induced by fatty acids and modified by hormones.     

Aldehyde dehydrogenase heterogeneity in rat hepatic cells

In normal rat liver, aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3; ALDH) is found primarily in mitochondrial and microsomal fractions. During hepatocarcinogenesis, an additional tumor-associated aldehyde dehydrogenase (T-ALDH) is detectable in the cytosol of preneoplastic and neoplastic cells. We report here differences in the ALDH distribution pattern in different rat hepatoma cell lines compared to normal rat hepatocytes. Of the four basal ALDH enzymes, one mitochondrial ALDH and one microsomal ALDH account for 96% of total ALDH molecules detectable with our probes in normal hepatocytes. The other two mitochondrial and microsomal ALDH enzymes are only detectable in the appropriate subcellular fraction from large populations of cells. The tumor-associated ALDH is not detectable in normal hepatocytes. In addition to varying amounts of T-ALDH in the six different rat hepatoma cell lines examined, differences in the amounts of mitochondrial and microsomal ALDHs also occur in both high and low T-ALDH activity hepatoma cell lines. Each of five ALDH enzymes examined has a characteristic half-life varying from 45 min to 95 h.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Differential regulation of insulin-like growth factor-II receptors in rat hepatocytes and hepatoma cells

This study compares the regulation of IGF-II receptors in three rat hepatoma lines, HTC, H-35 and 5123tc, and primary rat hepatocytes. In all cell types [125I]IGF-II bound solely to a species of approximately 250 kDa. Cell surface IGF-II receptors in hepatoma cells had slightly lower affinities (1-2 liters/nmol) than in hepatocytes (4 liters/nmol), but slightly higher IGF-I cross-reactivity (2-4% compared to 1% in hepatocytes). In confluent cultures, the three hepatoma lines expressed 5- to 15-fold more cell-surface receptors per cell than hepatocytes. However, while hepatocyte receptors showed marked inverse density-dependence, increasing over 6-fold between dense (3 x 10(5) cells/3.8 cm2) and sparse (0.16 x 10(5) cells/3.8 cm2) cultures, receptors in all hepatoma lines remained at a constant high level regardless of culture density. These distinct regulatory patterns resemble those described for growth-related functions in hepatocytes and hepatoma cells, and are thus consistent with a role for IGF-II receptors in liver cell proliferation.     

Immortalization of normal liver functions in cell culture: rat hepatocyte-hepatoma cell hybrids expressing ornithine carbamoyltransferase activity.

Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver-specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H-ornithine into protein arginine, and can be selectively grown in arginine-free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue-specific phenotypes.     

Primary cultured murine hepatocytes but not hepatoma cells regulate the cell number through density-dependent cell death

The mechanisms by which hepatocytes regulate their cell numbers in culture have been examined. We found that when murine hepatocytes were cultured at an overconfluent stage, the number of viable cells were reduced to that of the confluent stage 48 h later by cell death. Cell death was accompanied by LDH release, and it was observed only in primary cultured hepatocytes but not in hepatoma cells. Genomic DNA analysis using electrophoresis showed that DNA fragmentation, a biochemical hallmark of apoptosis, was induced in superconfluent cultures of hepatocytes in a cell-density-dependent fashion, but not in pre-confluent cells. DNA fragmentation was rapidly induced 2 h after the beginning of the in vitro culture and continued up to 24 h later. Flow cytometry analysis demonstrated that the nuclei from the hepatocytes in a high density culture were condensed and that the DNA content was reduced. These data suggest that the mechanism of cell death is apoptosis. The DNA fragmentation seen in the high density hepatocyte culture was not observed in hepatoma cell lines. Moreover, apoptosis was induced in hepatocytes of MRL/lpr mice, suggesting that the Fas antigen was not involved in the apoptotic process. Apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, and by a calmodulin antagonist, W-7. Taken together, the results indicate that high density culture of murine hepatocytes though not hepatoma cells regulate their cell numbers by an apoptotic mechanism. The apoptosis is dependent on de novo protein synthesis and intracellular calcium metabolism.     

Comparative subcellular distribution of benzaldehyde and acetaldehyde dehydrogenase activities in two hepatoma cell lines and in normal hepatocytes.

The NAD- and NADP-dependent aldehyde dehydrogenase (ALDH) activities were evaluated in two rat hepatoma cell lines, namely the well-differentiated MH1C1 line and the less differentiated HTC line. Each activity was determined in parallel in isolated rat hepatocytes, for comparison. The aliphatic aldehyde acetaldehyde (ACA) and the aromatic aldehyde benzaldehyde (BA) were used as substrates. With the first substrate the ALDH activities found in the crude cytoplasmic extracts were lower in hepatoma cells than in normal hepatocytes, especially when measured with NADP as coenzyme (ACA/NADP). Otherwise, with benzaldehyde as substrate the NAD-dependent enzyme activity (BA/NAD) was increased about 9-fold in HTC cells over hepatocytes and decreased in MH1C1 cells, while the NADP-dependent (BA/NADP) activity was increased 38- and 2.5-fold in HTC and MH1C1 cell lines, respectively. Studies on the subcellular distribution of these enzyme activities showed that the activity measured with acetaldehyde and NAD (ACA/NAD) was almost equally distributed between the cytosol and the subcellular particles in the three cell populations, but the ACA/NADP activity was shifted towards the cytosolic compartment in hepatomas, especially in HTC cells. The BA/NAD and BA/NADP ALDH activities found in the organelles of hepatoma cells were markedly reduced in comparison with hepatocytes, in favour of the cytosol. The most striking difference between the normal and the transformed cells was the 94-fold increase over hepatocytes of the BA/NADP activity, found in the cytosolic fractions of HTC cells. MH1C1 cells showed a less pronounced (7.5-fold) enhancement of this tumour-associated specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.     

Investigation by amperometric methods of intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes in the presence of different inhibitors of cellular metabolism

The reduction of 2,6-dichlorophenolindophenol (DCIP) was measured by amperometric methods in Morris hepatoma 3924A cells, normal isolated rat hepatocytes and in mitochondria isolated from normal rat liver. The influence of aerobic and anaerobic atmospheres and of various inhibitors of cellular metabolism, especially of the respiratory chain (KCN, NaN3, oligomycin), on DCIP-reduction were studied using glucose, succinate, beta-hydroxybutyrate, alpha-keto-glutarate and oxalacetate as substrates. Under the influence of KCN and oligomycin the velocity of DCIP-reduction was increased in both cell types. Azide showed a similar effect on tumour cells and to a lower extent on hepatocytes. Using isolated mitochondria total DCIPred was increased by KCN and azide using various mitochondrial metabolites as substrates and with ADP/Pi present. The effects of KCN, azide and oligomycin could be explained by taking DCIP as an artificial coupling site in mitochondria which is only used when oxygen is absent or when the respiratory chain is blocked by inhibitors of cytochrome oxidase. Evaluation of the reaction kinetics revealed differences between normal and transformed cells in terms of the pseudo-first-order rate constants and the activity of overall oxidoreductases. The results apparently reflect quantitative differences in enzymatic equipment and the metabolic pathways predominating in normal and neoplastic cells.     

Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture.

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLC/PRF/5, and SK-Hep-1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3-L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100 degrees C, with protease, with 2-mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cell also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.     

Characterization of the bile acid transport system in normal and transformed hepatocytes. Photoaffinity labeling of the taurocholate carrier protein

The taurocholate transport system in normal and transformed hepatocytes has been characterized using transport kinetics and photoaffinity labeling procedures. A photoreactive diazirine derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino [ 1,2-3H ]ethanesulfonic acid (7-ADTC), which has been shown to be a substrate for the bile acid carrier system, was photolyzed in the presence of intact hepatocytes, hepatoma tissue culture (HTC) cells, and plasma membranes derived from the hepatocyte sinusoidal surface. Irradiation of membranes in the presence of 7-ADTC resulted in the incorporation of the photoprobe into two proteins with Mr = 68,000 and 54,000. The specificity of labeling was confirmed by the significant inhibition of labeling observed when photolysis was carried out in the presence of taurocholate. The 68,000-Da protein was easily extracted with water and was shown to exhibit electrophoretic properties identical with rat serum albumin. The 54,000-Da protein required Triton X-100 for solubilization, indicating a strong association with the plasma membrane. Labeling of intact hepatocytes also resulted in specific labeling of the 54,000-Da protein. In contrast to hepatocytes, HTC cells derived from Morris hepatoma 7288C as well as H4-II-E cells derived from Reuber hepatoma H-35 exhibited a total loss of mediated bile acid uptake. Photolysis of 7-ADTC in the presence of HTC cells did not result in the labeling of any proteins, a result consistent with the loss of transport activity, and further supporting the specificity of the labeling reaction. The anion transport inhibitor N-(4-azido-2-nitrophenyl)-2-aminoethyl-[ 35S ]sulfonate, which has been shown to be a substrate for the bile acid carrier system also labeled the 54,000-Da plasma membrane protein when photolyzed in the presence of intact hepatocytes. These results suggest that the 54,000-Da protein is a component of the hepatocyte bile acid transport system and that the activity of this system is greatly reduced in several hepatoma cell lines.     

Essential role of intracellular glutathione in controlling ascorbic acid transporter expression and function in rat hepatocytes and hepatoma cells

Although there is in vivo evidence suggesting a role for glutathione in the metabolism and tissue distribution of vitamin C, no connection with the vitamin C transport systems has been reported. We show here that disruption of glutathione metabolism with buthionine-(S,R)-sulfoximine (BSO) produced a sustained blockade of ascorbic acid transport in rat hepatocytes and rat hepatoma cells. Rat hepatocytes expressed the Na(+)-coupled ascorbic acid transporter-1 (SVCT1), while hepatoma cells expressed the transporters SVCT1 and SVCT2. BSO-treated rat hepatoma cells showed a two order of magnitude decrease in SVCT1 and SVCT2 mRNA levels, undetectable SVCT1 and SVCT2 protein expression, and lacked the capacity to transport ascorbic acid, effects that were fully reversible on glutathione repletion. Interestingly, although SVCT1 mRNA levels remained unchanged in rat hepatocytes made glutathione deficient by in vivo BSO treatment, SVCT1 protein was absent from the plasma membrane and the cells lacked the capacity to transport ascorbic acid. The specificity of the BSO treatment was indicated by the finding that transport of oxidized vitamin C (dehydroascorbic acid) and glucose transporter expression were unaffected by BSO treatment. Moreover, glutathione depletion failed to affect ascorbic acid transport, and SVCT1 and SVCT2 expression in human hepatoma cells. Therefore, our data indicate an essential role for glutathione in controlling vitamin C metabolism in rat hepatocytes and rat hepatoma cells, two cell types capable of synthesizing ascorbic acid, by regulating the expression and subcellular localization of the transporters involved in the acquisition of ascorbic acid from extracellular sources, an effect not observed in human cells incapable of synthesizing ascorbic acid.     

Characterization of lipid and lipoprotein metabolism in primary human hepatocytes

Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model.     

Glycerol-3-phosphate dehydrogenase is induced by glucocorticoids in hepatocytes and hepatoma cells in vitro

Previous studies have shown that cytosolic glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) can be induced by glucocorticoids in mammalian brain, mammary gland, and thymus, but it was thought that no induction occurred in liver. We report here that GPDH is induced by glucocorticoids in several lines of hepatoma cells and in rat hepatocytes cultured in vitro. When rat hepatoma cells of clone FU5AH were exposed to 3 μM hydrocortisone (HC) for 3 days, GPDH specific activity increased greater than sixfold over control. The rate and extent of induction were similar in exponentially growing and stationary-phase cultures of cells. Four other hepatoma cell lines were inducible to a lesser extent, and three lines were not inducible. GPDH was also induced by glucocorticoids in cultures of hepatocytes isolated from livers of 6-day-old rats. The enzyme was induced threeto fourfold by the synthetic glucocorticoid, dexamethasone, in the presence of 1 nM insulin, but the induction was not observed in the absence of insulin.     

Establishment of a clonal strain of hepatoma cells which maintain in culture the five enzymes of the urea cycle

A clonal strain of epithelial cells has been established from the transplantable Morris hepatoma 7800 and is designated 7800C₁. The cells grow with a population doubling time of about three days in serum-supplemented synthetic medium. Cells of the 7800C₁ strain have maintained measurable activities of all the enzymes of the urea cycle during 17 months in continuous culture. The activity of argininosuccinate lyase is approximately that found in normal rat liver, while argininosuccinate synthetase, carbamoyl phosphate synthetase, arginase and ornithine carbamoyl transferase activities are, respectively, 40%, 28%, 6%. and 1% of normal values. Treatment of 7800C₁ cells with glucagon, dibutyryl 3′,5′-cyclic adenosine monophosphate or hydrocortisone did not increase the activity of any of the five enzymes.     

Interrelationship between sodium cyanate and pH in the regulation of tumor cell division

Previous studies have suggested that the selective inhibitory effects of sodium cyanate on tumor metabolism in vivo may be related to a lower interstitial pH in tumors. In the present work, the influence of extracellular pH on the actions of sodium cyanate was studied with one rat hepatoma cell line (HTC) and two human colon tumor cell lines (HT29 and LS174T) and with rat hepatocytes to determine if the effects are accompanied by changes in intracellular pH. With some tumor cells, an inhibition of cell proliferation was observed when the cells were exposed to an acidic medium (pH 6.6). However, the LS174T line of human tumor cells divided at pH 6.6 essentially as fast as at pH 7.4. In the concentration range of 0.02-0.1 mg/ml, a greater inhibitory effect of cyanate on cell proliferation was observed at the lower pH. Intracellular pH was found to be influenced by the sodium ion concentration of the medium to a similar degree in the three tumor lines that were examined. The intracellular pH was found to be significantly affected by cyanate in rat hepatocytes and in two of the tumor cell lines (HT29 and LS174T). The data suggested that not only does extracellular pH influence the inhibitory effect of cyanate on tumor cell proliferation but also that cyanate can affect the regulation of intracellular pH in normal and neoplastic cells.     

Adiponectin represses gluconeogenesis independent of insulin in hepatocytes

Adiponectin plays important roles in regulating insulin sensitivity and atherogenesis. Adiponectin has been shown to suppress hepatic glucose production in rodents. It has not been reported whether ectopically expressed adiponectin could regulate glucose metabolism in cultured hepatocyte-like cells. In the current study, the effect of adiponectin on glucose production was analyzed by ectopically expressing the protein in hepatoma H4IIE cells using an adenovirus delivery system to generate both human full-length and the globular domain of the protein. Expression of adiponectin in hepatoma H4IIE cells, in the absence of insulin, suppressed expression of the genes encoding glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, rate-limiting enzymes in the gluconeogenic pathway. Furthermore, expression of adiponectin in H4IIE cells suppressed glucose production from lactate and pyruvate. Purified recombinant human adiponectin also reduced glucose production in H4IIE cells and in rat primary hepatocytes in the absence of insulin. These data suggest that adiponectin protein could exert its function independent of the presence of insulin in these culture systems.     

Vitamin K uptake in hepatocytes and hepatoma cells

Hepatocellular carcinoma (HCC) or hepatoma cells have impaired ability to perform vitamin K-dependent carboxylation reactions. Vitamin K can also inhibit growth of HCC cells in vitro. Both carboxylation and growth inhibition are vitamin K dose dependent. We used rat hepatocytes, a vitamin K-growth sensitive (MH7777) and a vitamin K-growth resistant (H4IIE) rat hepatoma cell line to examine vitamin K uptake and vitamin K-mediated microsomal carboxylation. We found that vitamin K is taken up by normal rat hepatocytes against a saturable concentration gradient. The relative rates of uptake by rat hepatocytes and the two rat cell lines MH7777 and H4IIE correlated with their sensitivity to vitamin K-mediated cell growth inhibition. Pooled hepatocytes from liver nodules from rats treated with the hepatocarcinogen diethylnitrosamine (DEN) also had a reduced rate of vitamin K uptake. However, using a cell-free system, microsomes from both normal rat hepatocytes and the two rat hepatoma cell lines had a similar ability to support carboxylation mediated by exogenously added vitamin K. The results support the hypothesis that different sensitivity of hepatoma cells to vitamin K may be due to differences in vitamin K uptake and may be unrelated to the actions of vitamin K on carboxylation.