Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

In the process of isolated single liver cells coming together to form three-dimensional spheroids, cells undergo dramatic environmental changes. How liver cells respond to these changes has not been well studied before. This study characterized the functional and biochemical changes during liver spheroid formation and maintenance. Spheroids were prepared in 6-well plates from freshly isolated liver cells from male Sprague rats by a gyrotatory-mediated method. Morphological formation, and functional and biochemical parameters of liver spheroids were evaluated over a period of 21 days in culture. Liver spheroid formation was divided into two stages, immature (1-5 days) and mature (>5 days), according to their size and shape, and changes in their functionality. Galactose and pyruvate consumption was maintained at a relatively stable level throughout the period of observation. However, glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, decreased upto day 5 and remained stable thereafter. Cellular gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) activities were initially undetectable or low and increased as spheroids matured. Albumin secretion decreased rapidly within the first 2 days and increased as spheroids matured. It is concluded that cells undergo functional and biochemical changes during spheroid formation following isolation of liver cells from intact tissue. Functionality and biochemical properties recovered and were maintained in mature spheroids. A relatively stable period (6-15 days) of functionality in mature spheroids was identified and is recommended for applications of the model.     

Direct Self-assembly of Hepatocytes Spheroids within Hollow Fibers in Presence of Collagen

Opposite to the established view that collagen is an extracellular substratum for only dispersed hepatocyte culture, hepatocyte spheroids were directly formed within hollow fibers by addition of moderate concentrations of soluble collagen. Morphologically, these spheroids indicated a close relationship with their in vivo structure of liver. The albumin and urea synthetic profiles confirmed that those spheroids maintained liver-specific functions for at least 8 days. Spheroid formation by addition of collagen not only presents a potential methodology for clinical use of spheroids in bioartificial liver device but also indicates a likely function of collagen for self-assembly of primary cells in tissue engineering. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 25 November 2005; Accepted 25 November 2005     

Formation and characterization of three dimensional human hepatocyte cell line spheroids on chitosan matrix for in vitro tissue engineering applications

Chitosan was used as a matrix to induce three-dimensional spheroids of HepG2 cells. Chitosan films were prepared and used for culturing Hep G2 cells. Attachment kinetics of the cells was studied on the chitosan films. The optimum seeding density of the Hep G2 cells, required for three-dimensional spheroid formation was determined and was found to be 5 × 10⁴/ml. The growth kinetics of Hep G2 cells was studied using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, and morphology of the cells was studied through optical photographs taken at various days of culture. The liver cell functions of the spheroids were determined by measuring albumin and urea secretions. The results obtained from these studies have shown that the culture of Hep G2 cells on chitosan matrix taking appropriate seeding density resulted in the formation of three-dimensional spheroids and exhibited higher amount of albumin and urea synthesis compared to monolayer culture. These miniature “liver tissue like” models can be used for in vitro tissue engineering applications like preliminary evaluation of the toxicity of drugs and chemicals.     

Long-term culture of rat liver cell spheroids in hormonally defined media

Liver cells of new-born rats, which were found to be able to form spheroidal aggregates when cultured on a nonadherent plastic substratum, were studied under various conditions of culture, mainly by adding different nutrients and growth factors to the culture medium. Analysis of hepatocyte-specific functions was carried out by immunoprecipitation to detect specific proteins newly secreted by liver cell spheroids on different days of culture. When no supplement was added to culture medium, the secretion of albumin and transferrin by liver cell spheroids was no longer detectable after 2 weeks of culture. When dexamethasone, glucagon, insulin, and EGF were added to culture medium, the secretion of albumin and transferrin remained detectable at least until 60 days of culture. This was even more striking when trace elements were added in addition to the three hormones and EGF. The effects of addition of these various factors to culture medium were also detectable with respect to alpha-FP secretion. Even after 54 days of culture in total supplemented medium, these liver cell spheroids could be transferred on a collagen-coated plastic substratum to form a monolayer of uniform liver parenchyma-like cells. The presence of extracellular matrix-like material was observed on the surface of cell spheroids. This could be responsible for attachment and fusion between cell spheroids. Thus, liver cell spheroids cultured in total supplemented medium ensured cell attachment to a biological matrix and cell-cell contact, which is thought to help maintain cell differentiation. Liver cell spheroids offer the possibility of toxicological and pharmacological studies as well as cultures in biomatrix and coculture systems. In addition these liver cells can be used for experiments in liver cell transplantation.     

Inhibition of arginine synthesis by urea: A mechanism for arginine deficiency in renal failure which leads to increased hydroxyl radical generation

We have reported that (1) the synthesis of GSA, a uremic toxin, increases depending on the urea concentration and (2) GSA is formed from argininosuccinic acid (ASA) and the hydroxyl radical or SIN-1 which generates superoxide and NO simultaneously. However, an excess of NO, which also serves as a scavenger of the hydroxyl radical, inhibited GSA synthesis. We also reported that arginine, citrulline or ammonia plus ornithine, all of which increase arginine, inhibit GSA synthesis even in the presence of urea. To elucidate the mechanism for increased GSA synthesis by urea, we investigated the effect of urea on ASA and arginine, the immediate precursor of NO.Isolated rat hepatocytes were incubated in 6 ml of Krebs-Henseleit bicarbonate buffer containing 3% bovine serum albumin, 10 mM sodium lactate, 10 mM ammonium chloride and with or without 36 mM of urea and 0.5 or 5 mM ornithine at 37°C for 20 min. In vivo experiments, 4 ml/100 g body weight of 1.7 M urea or 1.7 M NaCl were injected intra-peritoneally into 5 male Wistar rats. Two hours after the intra-peritoneal injection of urea or 1.7 M NaCl, blood, liver and kidney were obtained by the freeze cramp method and amino acids were determined by an amino acid analyzer (JEOL:JCL-300).ASA in isolated hepatocytes was not detected with or without 36 mM (200 mgN/dl) urea, but the arginine level decreased from 36 to 33 nmol/g wet cells with urea. Ornithine which inhibits GSA synthesis, increased ASA markedly in a dose dependent manner and increased arginine. At 2 h after the urea injection the rat serum arginine level decreased by 42% (n = 5), and ornithine and citrulline levels increased significantly. Urea injection increased the ASA level in liver from 36–51 nmol/g liver but this was not statistically significant.We propose that urea inhibits arginine synthesis in hepatocytes, where the arginine level is extremely low to begin with, which decreases NO production which, in turn, increases hydroxyl radical generation from superoxide and NO. This may, also, be an explanation for the reported increase in oxygen stress in renal failure.     

In vitro formation of fibrous septa by liver connective tissue cells

Summary Active fibrous septa are a common feature in liver fibrosis and cirrhosis. Their etiology and formation were studied using cultures of tissue fragments or cells included in collagen gels. Liver fragments obtained from patients with cirrhosis or severe schistosomal fibrosis were able to reorganize the gel and to form discrete, interconnecting fibrous septa composed of parallel arrays of collagen, subsequently colonized by migrating connective tissue cells. The same was obtained in cultures of fibrogranulomatous lesions isolated from schistosome-infected mice livers. However, fragments of normal human and murine liver tissue did not show the capacity to form fibrous septa. Septa formation was also obtained in cultures of cell spheroids formed by liver connective tissue cells isolated from human fibrotic or cirrhotic liver tissues, but not with spheroids of normal skin fibroblasts or smooth muscle cells. This experimental model may represent the fibrous septa formation in vivo, depending on the activity of liver connective tissue cells. The ability of tissue fragments or cell spheroids to form septa in collagen gels might reflect the degree of fibrosis present in the liver tissue in vivo. This research was supported by FINEP and CNPq (Brazil) and CNRS (France).     

Development of liver microtissues with functional biliary ductular network

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.     

Extended liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gel

The potential use of porcine hepatocytes in a bioartificial liver device requires large quantities of viable and highly active cells. To facilitate the scaling up of the system, liver specific activities of hepatocytes should be maximized. One way of enhancing the specific activities is to cultivate hepatocytes as multicellular spheroids. Freshly isolated porcine hepatocytes form spheroids when cultivated in suspended cultures. These spheroids exhibit higher activities for a number of liver specific functions compared to hepatocytes cultivated as monolayers. However, these activities decreased in a few days in culture. Entrappment of spheroids in collagen gel sustained their metabolic activities at a stable level over 21 days. Production of albumin and urea by spheroid hepatocytes entrapped in collagen gels were 2 to 3 times higher than those by freshly isolated single cells. P-450 activity was demonstrated by metabolism of lidocaine to its main metabolite, monoethylglycinexylidide. Phase II drug metabolism was demonstrated by glucuronidation of 4-methylumbelliferone. This work shows that porcine hepatocyte spheroids entrapped in collagen maintain differentiated functions for an extended time period. Such hepatocyte spheroid entrappment system may facilitate the development of a bioartificial liver support device.     

Effect of arginine on the arginase activity and urea content of the brain and liver of rats acclimating to cold

At cold stress (3 days exposition at 2--4 degrees C) the urea formation in rats brain and liver does not become more active, the content of extraerythrocytic hemoglobin and the total peroxydase activity increase in blood serum, the animals sensitivity to the action of hyperbaric oxygenation (HB0) grows. At cold adaptation (45 days at 2--4 degrees C) the urea content in tissues and the activity of arginase in liver increase, the concentration of extraerythrocytic hemoglobin and the total peroxydase activity normalize, animals become more resistant to HB0. Every day administration of arginine during 3-day cold effect makes the brain and liver arginase on 42 and 28% more active, increases the urea content on 26 and 19%, stabilizes the erythrocytic membranes. The animals protected by arginine against cold are more resistant to the action of HB0.     

A role for PPARα in the regulation of arginine metabolism and nitric oxide synthesis

The pleiotropic effects of PPARα may include the regulation of amino acid metabolism. Nitric oxide (NO) is a key player in vascular homeostasis. NO synthesis may be jeopardized by a differential channeling of arginine toward urea (via arginase) versus NO (via NO synthase, NOS). This was studied in wild-type (WT) and PPARα-null (KO) mice fed diets containing either saturated fatty acids (COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers of arginine metabolism were assayed in urine and plasma. mRNA levels of arginases and NOS were determined in liver. Whole-body NO synthesis and the conversion of systemic arginine into urea were assessed by using ¹⁵N₂-guanido-arginine and measuring urinary ¹⁵NO₃ and [¹⁵N]-urea. PPARα deficiency resulted in a markedly lower whole-body NO synthesis, whereas the conversion of systemic arginine into urea remained unaffected. PPARα deficiency also increased plasma arginine and decreased citrulline concentration in plasma. These changes could not be ascribed to a direct effect on hepatic target genes, since NOS mRNA levels were unaffected, and arginase mRNA levels decreased in KO mice. Despite the low level in the diet, the nature of the fatty acids modulated some effects of PPARα deficiency, including plasma arginine and urea, which increased more in KO mice fed the LIN diet than in those fed the COCO diet. In conclusion, PPARα is largely involved in normal whole-body NO synthesis. This warrants further study on the potential of PPARα activation to maintain NO synthesis in the initiation of the metabolic syndrome.     

The effect of arginine and its metabolites on tissue culture of rat myocardium

In was demonstrated using the method of organotypic culture that arginine and its metabolites: cutrulline, ornitine and especially urea, in ultralow doses stimulated the myocardium proliferation in mature rats. Only nitric oxide (NO) possessed the antiproliferative effect. The cytrulline effect was similar to the arginine effect. The metabolism of these amino acids is intimately interrelated so the cytrulline can be used in the clinic practice as an "argininepreserved" preparation. The antiproliferative NO effect can be delayed by thiole-containing preparations: glulathione and unithiole.     

Inhibition of inducible nitric oxide synthase in murine visceral larva migrans: effects on lung and liver damage

The roles of nitric oxide production and oxidative process were studied in mice infected with Toxocara canis and treated with aminoguanidine which is a specific inhibitor of inducible nitric oxide synthase (iNOS). Relations of nitric oxide synthase inhibition and tissue pathology were assessed by biochemical, histological and immunohistochemical methods. In experiments, Balb/c albino mice were inoculated with T. canis eggs either with or without aminoguanidine treatment or alone, at 24th, 48th hours and on 7th days. LPx and SOD values in liver tissue and plasma were measured. Liver and lung tissues were evaluated for the pathological lesions. The expression of eNOS and iNOS in both tissues were studied with immunohistochemistry in the same intervals. We observed significant differences between T. canis infected and aminoguanidine treated animals. Larval toxocarosis led to oxidative stress elevation in plasma. Microscopic examination of the liver histological sections revealed pathological lesions in the hepatic parenchyma in infected mice. In the mice received T. canis eggs plus aminoguanidine, the sinusoidal areas were enlarged. Histological lesions were more severe at 48 hours after infection. Numbers of eNOS and iNOS expressing epithelial cells were increased in the T. canis infected mice. The activities of eNOS and iNOS were also observed in the body of the larvae which have migrated to lung and liver. As a result, we have demonstrated that in vivo production of eNO and iNO during T. canis infection cause direct host damages and it is strongly related to the oxidative stress. We propose that larval NO can also be effective in larval migration, but it needs further investigation on distribution of NO in larvae.     

Regulation of urea synthesis by agmatine in the perfused liver: studies with 15N

Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.     

Biochemical changes in blood at lead intoxication and pectin correction

The results of changes in liver biochemical parameters (AST, ALT, alkaline phosphatase, total protein, urea, and bilirubin) indicating the presence and type of liver injury in lead intoxication and subsequent correction with pectin.     

Urea formation by postoperative liver

The effect of liver left lobe resection (LR, 15–20% of the organ weight) on hepatic urea formation was investigated in 84 albino rats. The objects used for analysis of urea content included: operated left lobe (LLL), non-operated middle (MLL) lobe of the liver, blood (aorta, v. hepatica, v. porta), and choledochal bile. Arginase activity was examined in liver homogenate. On the day 3 and day 7 after resection reduced arginase activity was detected. LR caused a decrease in urea content in v. hepatica, but increased urea content in the arterial blood and v. porta. The increase in bile urea on day 7 was then changed for its decrease observed on day 14 of the postoperative period. The urea content in the liver on day 3 after LR was below the norm, while on days 7 and 14 it became normal. Results of this study suggest impaired urea formation by hepatocytes of the operated liver and activation of extrahepatic mechanisms of urea formation from arginine.     

Metabolic and mitochondrial disturbances in streptozotocin-treated Sprague-Dawley and Sherman rats

This study provides explanation for conflicting evidence in the literature relating to changes in mitochondrial function and metabolic parameters during chemically induced diabetes. Diabetes of 3 days' duration (early ketosis) did not alter heart, kidney, or liver mitochondrial respiratory rates with glutamate or succinate even though serum glucose and triglycerides were elevated. Diabetes of 5 weeks' duration did not alter kidney or liver mitochondrial function in the fed adult rat although weight gain was depressed. The amount of kidney mitochondrial protein isolated per gram of tissue was increased by 30% in the diabetic. This increase was reversed by insulin treatment as were the other biochemical modalities measured. Superimposition of a 24-hr fast resulted in enhanced gluconeogenesis as measured by an animal weight loss of 17% within 24 hr (liver weight loss, 21%) and an elevation of serum urea nitrogen by 180% compared to fasted control. Respiratory rates of diabetic kidney mitochondria with glutamate were unaffected in the fasted animal whereas diabetic liver mitochondrial respiratory rates during succinate oxidation were reduced by 43%. Respiratory control was unchanged in the fasted diabetic rat. All the observed changes were reversed by insulin. Variation in the serum and liver metabolic indices (urea nitrogen, creatinine, glycerol, free fatty acids, free amino acids, triglycerides, and glucose) and liver mitochondrial responses to 7 weeks of chemically induced diabetes was affected by the rat strain, Sprague-Dawley versus Sherman, and rat weight, 72 g versus 222 g. Liver mitochondrial respirations in fed Sherman rats were not depressed by diabetes. Both rat strains had elevated liver free fatty acids and glutamate dehydrogenase activity in the diabetic state. Serum leucine, isoleucine, and valine were more elevated and serum lysine and arginine were more depressed in the diabetic Sprague-Dawley rat than in the Sherman rat. Conjectures on these results are presented in the text.     

Transcriptome atlas of glutamine family amino acid metabolism‐related genes in eight regenerating liver cell types

To explore glutamine family amino acid metabolism of eight liver cell types in rat liver regeneration, eight kinds of rat regenerating liver cells were isolated by using the combination of Percoll density gradient centrifugation and immunomagnetic bead methods, then Rat Genome 230 2.0 Array was used to detect the expression profiles of the genes associated with metabolism of glutamine family amino acid in rat liver regeneration and finally how these genes involved in activities of eight regenerating liver cell types were analysed by the methods of bioinformatics and systems biology. The results showed that in the priming stage of liver regeneration, hepatic stellate cells and sinusoidal endothelial cells transformed proline and glutamine into glutamate; hepatocytes, hepatic stellate cells, sinusoidal endothelial cells and dendritic cells catabolized glutamate to 2‐oxoglutarate or succinate; hepatic stellate cells and sinusoidal endothelial cells catalysed glutamate into glutamyl‐tRNA for protein synthesis; urea cycle, which degraded from arginine, was enhanced in biliary epithelia cells, sinusoidal endothelial cells and dendritic cells; synthesis of polyamines from arginine was enhanced in biliary epithelia cells, sinusoidal endothelial cells, Kupffer cells and dendritic cells; the content of NO was increased in sinusoidal endothelial cells and dendritic cells; degradation of proline was enhanced in hepatocytes and biliary epithelia cells. In the progress stage, biliary epithelia cells converted glutamine into GMP and glucosamine 6‐phosphate; oval cells converted glutamine into glucosamine 6‐phosphate; hepatic stellate cells converted glutamine into NAD; the content of NO, which degraded from arginine, was increased in biliary epithelia cells, oval cells, pit cells and dendritic cells. In the termination stage, oval cells converted proline into glutamate; glutamate degradation, which degraded from arginine, was enhanced in hepatocytes and dendritic cells; the content of NO was increased in oval cells, sinusoidal endothelial cells, pit cells and dendritic cells. The synthesis of creatine phosphate was enhanced in hepatocytes, biliary epithelia cells, pit cells and dendritic cells in both progress and termination stages. In summary, glutamine family amino acid metabolism has some differences in liver regeneration in different liver cells.     

Effects of dietary salt intake on plasma arginine

Because L-arginine is degraded by hepatic arginase to ornithine and urea and is transported by the regulated 2A cationic amino acid y(+) transporter (CAT2A), hepatic transport may regulate plasma arginine concentration. Groups of rats (n = 6) were fed a diet of either low salt (LS) or high salt (HS) for 7 days to test the hypothesis that dietary salt intake regulates plasma arginine concentration and renal nitric oxide (NO) generation by measuring plasma arginine and ornithine concentrations, renal NO excretion, and expression of hepatic CAT2A, and arginase. LS rats had lower excretion of NO metabolites and cGMP, lower plasma arginine concentration (LS: 83 +/- 7 vs. HS: 165 +/- 10 micromol/l, P < 0.001), but higher plasma ornithine concentration (LS: 82 +/- 6 vs. HS: 66 +/- 4 micromol/l, P < 0.05) and urea excretion. However, neither the in vitro hepatic arginase activity nor the mRNA for hepatic arginase I was different between groups. In contrast, LS rats had twice the abundance of mRNA for hepatic CAT2A (LS: 3.4 +/- 0.4 vs. HS: 1.6 +/- 0.5, P < 0.05). The reduced plasma arginine concentration with increased plasma ornithine concentration and urea excretion during LS indicates increased arginine metabolism by arginase. This cannot be ascribed to changes in hepatic arginase expression but may be a consequence of increased hepatic arginine uptake via CAT2A.     

Arginase expression and modulation of IL-1beta-induced nitric oxide generation in rat and human islets of Langerhans.

Proinflammatory cytokine induction of NO synthesis may contribute to the destruction of pancreatic beta cells leading to type 1 diabetes. The NO synthase substrate arginine can also be metabolized to ornithine and urea in a reaction catalyzed by cytosolic (AI) or mitochondrial (AII) isoforms of arginase. Recent evidence suggests that the rate of NO generation is dependent on the relative activities of NO synthase and arginase. The objectives of this study were (i). to identify the arginase isoforms expressed in rat and human islets of Langerhans and a rat beta cell line, RINm5F and (ii). to investigate the competition for arginine between NO synthase and arginase in IL-1beta-treated rat islets. Arginase activity was detected in rat islets (fresh tissue, 346 mU/mg protein; cultured, 587 mU/mg), cultured human islets (56 mU/mg), RINm5F cells (376 mU/mg), rat kidney (238 mU/mg), and rat liver (6119 mU/mg). Using Western blots, AI was shown to be the predominant isoform expressed in rat islets and in RINm5F cells while human islets expressed far more AII than AI. Rat islets were cultured in medium containing 1.14, 0.1, and 0.01 mM arginine and treated with IL-1beta and the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). IL-1beta-induced NO generation was unaffected by ABH at 1.14 mM arginine, but significantly increased at 0.1 and 0.01 mM arginine. These findings suggest that the level of islet arginase activity can regulate the rate of induced NO generation and this may be relevant to the insulitis process leading to beta cell destruction in type 1 diabetes.