Interorgan ammonia metabolism in liver failure

In the post-absorptive state, ammonia is produced in equal amounts in the small and large bowel. Small intestinal synthesis of ammonia is related to amino acid breakdown, whereas large bowel ammonia production is caused by bacterial breakdown of amino acids and urea. The contribution of the gut to the hyperammonemic state observed during liver failure is mainly due to portacaval shunting and not the result of changes in the metabolism of ammonia in the gut. Patients with liver disease have reduced urea synthesis capacity and reduced peri-venous glutamine synthesis capacity, resulting in reduced capacity to detoxify ammonia in the liver.The kidneys produce ammonia but adapt to liver failure in experimental portacaval shunting by reducing ammonia release into the systemic circulation. The kidneys have the ability to switch from net ammonia production to net ammonia excretion, which is beneficial for the hyperammonemic patient. Data in experimental animals suggest that the kidneys could have a major role in post-feeding and post-haemorrhagic hyperammonemia.During hyperammonemia, muscle takes up ammonia and plays a major role in (temporarily) detoxifying ammonia to glutamine. Net uptake of ammonia by the brain occurs in patients and experimental animals with acute and chronic liver failure. Concomitant release of glutamine has been demonstrated in experimental animals, together with large increases of the cerebral cortex ammonia and glutamine concentrations. In this review we will discuss interorgan trafficking of ammonia during acute and chronic liver failure. Interorgan glutamine metabolism is also briefly discussed, since glutamine synthesis from glutamate and ammonia is an important alternative pathway of ammonia detoxification. The main ammonia producing organs are the intestines and the kidneys, whereas the major ammonia consuming organs are the liver and the muscle.     

Hepatocyte heterogeneity in ammonia metabolism: impairment of glutamine synthesis in CCl4 induced liver cell necrosis with no effect on urea synthesis

After induction of a perivenous liver cell necrosis by CCl4 pretreatment of the rat, ammonia uptake by perfused liver is decreased. This was due to an inhibition of glutamine synthesis from added ammonia, whereas urea synthesis was not affected by CCl4 pretreatment. The data confirm recent findings on hepatocyte heterogeneity in ammonia metabolism and are explained by an impairment of perivenous glutamine synthetase, but not of periportal urea synthesis, by the perivenous liver cell necrosis induced by CCl4. Regarding the pathogenesis of hyperammonemia in acute severe liver disease like CCl4 poisoning, the data point to a role of an impaired glutamine synthesis, but not to an impairment of urea synthesis.     

Effect of Urease-Induced Hyperammonemia on Metabolism of Guanidino Compounds

We previously reported that guanidino compounds produced by the catabolism of arginine play an important role in the pathophysiology of acute hyperammonemia. In order to understand the metabolism of guanidino compounds during sustained hyperammonemia, we investigated the effect of intraperitoneal urease injection (800 IU/kg) on the levels of guanidino compounds in blood, liver, kidney, and brain of rats. Control rats received an equal volume of saline. Eight hours following injection, rats were sacrificed and blood and tissues were removed. Ammonia and urea were determined by enzymatic and colorimetric assays, respectively. Guanidino compounds were analyzed by high-performance liquid chromatography. Blood and tissue ammonia were significantly increased and urea decreased in urease-treated animals. Blood and kidney arginine levels were significantly decreased although hepatic arginine was increased following urease injection. Elevated hepatic arginine may be due to the rapid conversion of urea to ammonia by urease and the development of a futile urea cycle. Catabolites produced by the transamidination of arginine were significantly decreased in the blood, liver, kidney, and brain of urease-treated rats, whereas acetylation of hepatic arginine to α-N-acetylarginine was increased. Blood and tissue guanidinosuccinic acid levels were not elevated during urease induced hyperammonemia, supporting the hypothesis that urea is a precursor for the synthesis of guanidinosuccinic acid.     

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: Involvement of mitochondrial aquaporin-8

We recently reported that hepatocyte mitochondrial aquaporin-8 (mtAQP8) channels facilitate the uptake of ammonia and its metabolism into urea. Here we studied the effect of bacterial lipopolysaccharides (LPS) on ammonia-derived ureagenesis. In LPS-treated rats, hepatic mtAQP8 protein expression and diffusional ammonia permeability (measured utilizing ammonia analogues) of liver inner mitochondrial membranes were downregulated. NMR studies using 15 N-labeled ammonia indicated that basal and glucagon-induced ureagenesis from ammonia were significantly reduced in hepatocytes from LPS-treated rats. Our data suggest that hepatocyte mtAQP8-mediated ammonia removal via ureagenesis is impaired by LPS, a mechanism potentially relevant to the molecular pathogenesis of defective hepatic ammonia detoxification in sepsis.     

Hepatocyte heterogeneity in the metabolism of amino acids and ammonia.

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate synthase and glutamine synthase in the liver lobule. The mechanisms underlying the different sublobular gene expressions are not yet settled but may involve the development of hepatic architecture, innervation, blood-borne hormonal and metabolic factors. The periportal zone is characterized by a high capacity for uptake and catabolism of amino acids (except glutamate and aspartate) as well as for urea synthesis and gluconeogenesis. On the other hand, glutamine synthesis, ornithine transamination and the uptake of vascular glutamate, aspartate, malate and alpha-ketoglutarate are restricted to a small perivenous hepatocyte population. Accordingly, in the intact liver lobule the major pathways for ammonia detoxication, urea and glutamine synthesis, are anatomically switched behind each other and represent in functional terms the sequence of the periportal low affinity system (urea synthesis) and a previous high affinity system (glutamine synthesis) for ammonia detoxication. Perivenous glutamine synthase-containing hepatocytes ('scavenger cells') act as a high affinity scavenger for the ammonia, which escapes the more upstream urea-synthesizing compartment. Periportal glutaminase acts as a pH- and hormone-modulated ammonia-amplifying system in the mitochondria of periportal hepatocytes. The activity of this amplifying system is one crucial determinant for flux through the urea cycle in view of the high Km (ammonia) of carbamoylphosphate synthase, the rate-controlling enzyme of the urea cycle. The structural and functional organization of glutamine and ammonia-metabolizing pathways in the liver lobule provides one basis for the understanding of a hepatic role in systemic acid base homeostasis. Urea synthesis is a major pathway for irreversible removal of metabolically generated bicarbonate. The lobular organization enables the adjustment of the urea cycle flux and accordingly the rate of irreversible hepatic bicarbonate elimination to the needs of the systemic acid base situation, without the threat of hyperammonemia.     

Metabolic pre-conditioning of cultured cells in physiological levels of insulin: generating resistance to the lipid-accumulating effects of plasma in hepatocytes

Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.     

High level benzodiazepine and ammonia clearance by flat membrane bioreactors with porcine liver cells

The onset of hepatic encephalopathy is a multifactorial process in which endogenous benzodiazepines and hyperammonemia play a pivotal role. The treatment of comatose states in liver failure is one of the major functions of a bioartificial liver. A controlled study demonstrating the capacity of a large scale bioartificial liver to detoxify benzodiazepines could be a crucial prerequisite to break this circle of events leading to coma. The aim of this study was therefore to expose the bioreactor to high levels of benzodiazepines and ammonia for evaluation of its detoxifying capacity. We have developed a novel and unique device reconstructing the plate architecture of the liver. Porcine hepatocytes were co-cultured with non-parenchymal cells. We investigated benzodiazepine metabolism using diazepam as model drug. The bioreactor was also loaded with high levels of ammonia and ammonia clearance as well as urea secretion with ammonia challenge were investigated. Albumin secretion was analysed in parallel as a control viability and tissue specific secretory parameter. The results clearly show that the velocity of diazepam turnover increases between day 1 and 2 and stabilises at high levels. Typical diazepam metabolites including temazepam, N-desmethyl-diazepam and oxazepam were generated. Cell specific functions, including albumin secretion, were comparable to an in vivo liver. We conclude that the flat membrane bioreactor used as bioartificial liver has the potential to detoxify diazepam and ammonia at significant amounts. Maintenance of monoxygenase activities in vitro is one of the strongholds of the bioreactor concept presented in this study.     

A novel full-scale flat membrane bioreactor utilizing porcine hepatocytes: cell viability and tissue-specific functions

When designing an extracorporeal hybrid liver support device, special attention should be paid to providing the architectural basis for reconstructing a proper cellular microenvironment that ensures highest and prolonged functional activity of the liver cells. The common goal is to achieve high cell density culture and to design the bioreactor for full-scale primary liver cell cultures under adequate mass transfer conditions. An important aim of this study was to evaluate the biochemical performance of a flat membrane bioreactor that permits high-density hepatocyte culture and simultaneously to culture cells under sufficient oxygenation availability conditions comparable to the in vivo-like microenvironment. In such a bioreactor pig liver cells were cultured within an extracellular matrix between oxygen-permeable flat-sheet membranes. In this investigation we used a novel scaled-up prototype consisting of up to 20 modules in a parallel mode. Each module was seeded with 2 x 10(8) cells. Microscopic examination of the hepatocytes revealed morphological characteristics as found in vivo. Cell concentration increased in the first days of culture, as indicated by DNA measurements. The performance of the bioreactor was monitored for 18 days in terms of albumin synthesis, urea synthesis, ammonia elimination, and diazepam metabolism. The ability of the hepatocytes to synthesize albumin and urea increased during the first days of culture. Higher rates of albumin synthesis were obtained at day 9 and remained at a value of 1.41 pg/h/cell until day 18 of culture. The rate of urea synthesis increased from 23 ng/h/cell to 28 ng/h/cell and then remained constant. Cells eliminated ammonia at a rate of about 56 pg/h/cell, which was constant over the experimental period. Hepatocytes in the bioreactor metabolized diazepam and generated three different metabolites: nordiazepam, temazepam, and oxazepam. The production of such metabolites was sustained until 18 days of culture. These results demonstrated that the scale-up of the bioreactor was assessed, and it could be demonstrated that the device design aimed at the reconstruction of the liver-specific tissue architecture supported the expression of liver-specific functions of primary pig liver cells.     

Ammonia removal using hepatoma cells in mammalian cell cultures

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.     

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.     

Urea-cycle disorders as a paradigm for inborn errors of hepatocyte metabolism

Urea-cycle disorders (UCDs) are a group of inborn errors of hepatocyte metabolism that are caused by the loss of enzymes involved in the process of transferring nitrogen from ammonia to urea, via the urea cycle (UC). Recent genetic analyses of inherited disorders that present with hyperammonemia demonstrate the function of cellular transporters that regulate the availability of UC intermediates. The regulation of UC intermediates, such as arginine, could have far reaching implications on nitric-oxide synthesis and vascular tone. Hence, each UCD and UC-related disorder constitutes a unique gene-nutrient interaction that is crucial for postnatal homeostasis. Recent advances in the diagnosis and management of UCDs include the application of in vivo metabolic-flux measurements. Cumulative morbidity is still high despite dietary and pharmacological therapies and, hence, both cell and gene therapies are being pursued as possible long-term corrective treatments. Although gene-replacement therapy has suffered recent clinical setbacks, new vector developments offer hope for the treatment of cell-autonomous defects of hepatocyte metabolism.     

Interorgan ammonia, glutamate, and glutamine trafficking in pigs with acute liver failure

Ammonia reduction is the target for therapy of hepatic encephalopathy, but lack of quantitative data about how the individual organs handle ammonia limits our ability to develop novel therapeutic strategies. The study aims were to evaluate interorgan ammonia metabolism quantitatively in a devascularized pig model of acute liver failure (ALF). Ammonia and amino acid fluxes were measured across the portal drained viscera (PDV), kidneys, hind leg, and lungs in ALF pigs. ALF pigs developed hyperammonemia and increased glutamine levels, whereas glutamate levels were decreased. PDV contributed to the hyperammonemic state mainly through increased shunting and not as a result of increased glutamine breakdown. The kidneys were quantitatively as important as PDV in systemic ammonia release, whereas muscle took up ammonia. Data suggest that the lungs are able to remove ammonia from the circulation during the initial stage of ALF. Our study provides new data supporting the concept of glutamate deficiency in a pig model of ALF. Furthermore, the kidneys are quantitatively as important as PDV in ammonia production, and the muscles play an important role in ammonia removal.     

Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules.

The metabolism of cysteine and cysteinesulphinate was studied in freshly isolated rat hepatocytes. Over 80% of the 14CO2 formed from [1-14C]cysteinesulphinate could be accounted for by production of hypotaurine plus taurine in incubations of rat hepatocytes with either 1 mM- or 25 mM-cysteinesulphinate. In similar incubations with 1 mM- or 25 mM-cysteine, less than 10% of 14CO2 evolution from [1-14C]cysteine could be accounted for by production of hypotaurine plus taurine. In incubations with cysteine, but not with cysteinesulphinate, the production of urea and ammonia was substantially increased above that observed in incubations without substrate. Addition of unlabelled cysteinesulphinate did not affect 14CO2 production from [1-14C]cysteine. Addition of 2-oxoglutarate resulted in a marked increase in cysteinesulphinate catabolism via the transamination pathway, but addition of neither 2-oxoglutarate nor pyruvate to the incubation system had any effect on cysteine catabolism. Inhibition of cystathionase with propargylglycine decreased 14CO2 production from [1-14C]cysteine about 50% and markedly decreased production of ammonia plus urea N; cysteinesulphinate catabolism by cysteinesulphinate-independent pathways in the rat hepatocyte and, furthermore, that cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for cysteine catabolism in rat liver.     

Cerebral metabolic disturbances in the brain during acute liver failure: from hyperammonemia to energy failure and proteolysis

Several observations suggest that patients with fulminant hepatic failure may suffer from disturbances in cerebral metabolism that can be related to elevated levels of arterial ammonia. One effect of ammonia is the inhibition of the rate limiting TCA cycle enzyme alpha-ketoglutarate dehydrogenase (alphaKGDH) and possibly also pyruvate dehydrogenase, but this has been regarded to be of no quantitative importance. However, recent studies justify a revision of this point of view. Based on published data, the following sequence of events is proposed. Inhibition of alphaKGDH both enhances the detoxification of ammonia by formation of glutamine from alpha-ketoglutarate and reduces the rate of NADH and oxidative ATP production in astrocytic mitochondria. In the astrocytic cytosol this will lead to formation of lactate even in the presence of sufficient oxygen supply. Since the aspartate-malate shuttle is compromised, there is a risk of depletion of mitochondrial NADH and ATP unless compensatory mechanisms are recruited. One likely compensatory mechanism is the use of amino acids for energy production. Branched chain amino acids, like isoleucine and valine can supply carbon skeletons that bypass the alphaKGDH inhibition and maintain TCA cycle activity. Large-scale consumption of certain amino acids can only be maintained by cerebral proteolysis, as has been observed in these patients. This hypothesis provides a link between hyperammonemia, ammonia detoxification by glutamine production, cerebral lactate production, and cerebral catabolic proteolysis in patients with FHF.