Dietary fiber alleviates alcoholic liver injury via Bacteroides acidifaciens and subsequent ammonia detoxification.

The gut microbiota and diet-induced changes in microbiome composition have been linked to various liver diseases, although the specific microbes and mechanisms remain understudied. Alcohol-related liver disease (ALD) is one such disease with limited therapeutic options due to its complex pathogenesis. We demonstrate that a diet rich in soluble dietary fiber increases the abundance of Bacteroides acidifaciens (B. acidifaciens) and alleviates alcohol-induced liver injury in mice. B. acidifaciens treatment alone ameliorates liver injury through a bile salt hydrolase that generates unconjugated bile acids to activate intestinal farnesoid X receptor (FXR) and its downstream target, fibroblast growth factor-15 (FGF15). FGF15 promotes hepatocyte expression of ornithine aminotransferase (OAT), which facilitates the metabolism of accumulated ornithine in the liver into glutamate, thereby providing sufficient glutamate for ammonia detoxification via the glutamine synthesis pathway. Collectively, these findings uncover a potential therapeutic strategy for ALD involving dietary fiber supplementation and B. acidifaciens.

Quo vadis ureagenesis disorders? A journey from 90 years ago into the future.

The pathway of ammonia disposal in the mammalian organism has been described in 1932 as a metabolic cycle present in the liver in different compartments. In 1958, the first human disorder affecting this pathway was described as a genetic condition leading to cognitive impairment and constant abnormalities of amino acid metabolism. Since then, defects in all enzymes and transporters of the urea cycle have been described, referring to them as primary urea cycle disorders causing primary hyperammonemia. In addition, there is a still increasing list of conditions that impact on the function of the urea cycle by various mechanisms, hereby leading to secondary hyperammonemia. Despite great advances in understanding the molecular background and the biochemical specificities of both primary and secondary hyperammonemias, there remain many open questions: we do not fully understand the pathophysiology in many of the conditions; we do not always understand the highly variable clinical course of affected patients; we clearly appreciate the need for novel and improved diagnostic and therapeutic approaches. This study does look back to the beginning of the urea cycle (hi)story, briefly describes the journey through past decades, hereby illustrating advancements and knowledge gaps, and gives examples for the extremely broad perspective imminent to some of the defects of ureagenesis and allied conditions.

The role of the balance between energy production and ammonia detoxification mediated by key amino acids in divergent hypersaline adaptation among crassostrea oysters.

Global ocean salinity is changing under rapid climate change and intensified anthropogenic activity. Increased differences in salinity threaten marine biodiversity, organismal survival, and evolution, particularly sessile invertebrates dwelling in highly fluctuating intertidal and estuarine environments. Comparing the responses of closely related species to salinity changes can provide insights into the adaptive mechanisms underlying inter- and intraspecific divergence in salinity tolerance, but are poorly understood in marine bivalves. We collected wild individuals of four Crassostrea species, in addition to two populations of the same species from their native habitats and determined the dynamics of hydrolyzed amino acids (HAAs) and transcriptional responses to hypersaline stress. In response to hypersaline stress, species/populations inhabiting natural high-salinity sea environments showed higher survival and less decline in HAAs than that of congeners inhabiting low-salinity estuaries. Thus, native environmental salinity shapes oyster tolerance. Notably, a strong negative correlation between the decline in HAAs and survival indicated that the HAAs pool could predict tolerance to hypersaline challenge. Four HAAs, including glutamine (Glu), aspartic acid (Asp), alanine (Ala) and glycine (Gly), were identified as key amino acids that contributed substantially to the emergency response to hypersaline stress. High-salinity-adapted oyster species only induced substantial decreases in Glu and Asp, whereas low-salinity-adapted congeners further incresaed Ala and Gly metabolism under hypersaline stress. The dynamics of the content and gene expression responsible for key amino acids pathways revealed the importance of maintaining the balance between energy production and ammonia detoxification in divergent hypersaline responses among oyster species/populations. High constructive or plastic expression of evolutionarily expanded gene copies in high-salinity-adapted species may contribute to their greater hypersaline tolerance. Our findings reveal the adaptive mechanism of key amino acids in salinity adaptation in marine bivalves and provide new avenues for the prediction of adaptive potential and aquaculture with high-salinity tolerant germplasms.

Sericin enhances ammonia detoxification by promotes urea cycle enzyme genes and activates hepatic autophagy in relation to CARD-9/MAPK pathway.

Urea cycle is an important metabolic process that initiates in liver mitochondria and converts ammonia to urea. The impairment of ammonia detoxification, both primary and secondary causes, lead to hyperammonemia, a life-threatening condition affecting to the brain. Current treatments are not enough effective. In addition, our recent proteomics study in hypercholesterolemic rat model demonstrated that sericin enhances hepatic nitrogenous waste removal through carbamoyl-phosphate synthase 1 (CPS-1), aldehyde dehydrogenase-2 (ALDH-2), and uricase proteins. However, the underlining mechanisms regard to this property is not clarified yet. Therefore, the present study aims to examine the effect of sericin on urea cycle enzyme genes (CPS-1 and ornithine transcarbamylase; OTC) and proteins (mitogen-activated protein kinase; MAPK, caspase recruitment domain-containing protein 9; CARD-9, Microtubule-associated protein light chain 3; LC-3), which relate to urea production and liver homeostasis in hepatic cell line (HepG2) and hypercholesterolemic rat treated with or without sericin. qRT-PCR, immunohistochemistry, and electron microscopy techniques were performed. In vitro study determined that high dose of sericin at 1 mg/ml increased liver detoxification enzyme (Cytochrome P450 1A2; CYP1A2 and ALDH-2) and urea cycle enzyme (CPS-1 and OTC) genes. Both in HepG2 cell and rat liver mitochondria, sericin significantly downregulated CARD-9 (apoptotic protein) expression while upregulated MAPK (hepatic homeostasis protein) and LC-3 (autophagic protein) expressions. Hence, it might be concluded that sericin promotes ammonia detoxification by both increases urea cycle enzyme genes and enhances hepatic autophagy in associated with CARD-9/MAPK pathway (as shown by their own negative relationship). This study presents another beneficial property of sericin to develop an upcoming candidate for ammonia toxicity alleviation and liver function improvement.

The short-term effects of berberine in the liver: Narrow margins between benefits and toxicity.

Berberine is a plant alkaloid to which antihyperglycemic properties have been attributed. It is also known as an inhibitor of mitochondrial functions. In this work short-term translation of the latter effects on hepatic metabolism were investigated using the isolated perfused rat liver. Once-through perfusion with a buffered saline solution was done. At low portal concentrations berberine modified several metabolic pathways. It inhibited hepatic gluconeogenesis, increased glycolysis, inhibited ammonia detoxification, increased the cytosolic NADH/NAD+ ratio and diminished the ATP levels. Respiration of intact mitochondria was impaired as well as the mitochondrial pyruvate carboxylation activity. These results can be regarded as evidence that the direct inhibitory effects of berberine on gluconeogenesis, mediated by both energy metabolism and pyruvate carboxylation inhibition, represent most likely a significant contribution to its clinical efficacy as an antihyperglycemic agent. However, safety concerns also arise because all effects occur at similar concentrations and there is a narrow margin between the expected benefits and toxicity. Even mild inhibition of gluconeogenesis is accompanied by diminutions in oxygen uptake and ammonia detoxification and increases in the NADH/NAD+ ratio. All combined, desired and undesired effects could well in the end represent a deleterious combination of events leading to disruption of cellular homeostasis.

Astrocytic urea cycle detoxifies Aß-derived ammonia while impairing memory in Alzheimer's disease.

Alzheimer's disease (AD) is one of the foremost neurodegenerative diseases, characterized by beta-amyloid (Aß) plaques and significant progressive memory loss. In AD, astrocytes are proposed to take up and clear Aß plaques. However, how Aß induces pathogenesis and memory impairment in AD remains elusive. We report that normal astrocytes show non-cyclic urea metabolism, whereas Aß-treated astrocytes show switched-on urea cycle with upregulated enzymes and accumulated entering-metabolite aspartate, starting-substrate ammonia, end-product urea, and side-product putrescine. Gene silencing of astrocytic ornithine decarboxylase-1 (ODC1), facilitating ornithine-to-putrescine conversion, boosts urea cycle and eliminates aberrant putrescine and its toxic byproducts ammonia and H2O2 and its end product GABA to recover from reactive astrogliosis and memory impairment in AD. Our findings implicate that astrocytic urea cycle exerts opposing roles of beneficial Aß detoxification and detrimental memory impairment in AD. We propose ODC1 inhibition as a promising therapeutic strategy for AD to facilitate removal of toxic molecules and prevent memory loss.

Adverse effects of dietary virgin (nano)microplastics on growth performance, immune response, and resistance to ammonia stress and pathogen challenge in juvenile sea cucumber Apostichopus japonicus (Selenka).

It has been well documented that micro- and nanoplastics are emerging pollutants in aquatic environments, and their potential toxic effects has attracted widespread concerns. Here, we evaluated the adverse effects of dietary polystyrene nanoplastics and microplastics (PS-N/MPs) on growth performance, oxidative stress induction, immune response, ammonia detoxification, and bacterial pathogen resistance of sea cucumber Apostichopus japonicus. After collection and acclimation, sea cucumbers were randomized into 3 groups (i.e., control, 100 nm PS-NPs and 20 µm PS-MPs at 100 mg kg-1 diet) for 60-day feeding experiment. Every group contained 360 sea cucumbers which were equally divided into 3 aquaria as biological triplicates. The results showed that the specific growth rate and final weight of the sea cucumbers fed with diets containing PS-N/MPs were significantly lower than those of control group. Dietary virgin PS-N/MPs significantly increased the reactive oxygen species production and malondialdehyde content in coelomic fluid, causing oxidative stress and damage to the growth and development of A. japonicus. During the experiment, 100 nm PS-NPs significantly induced the depletion in cellular and humoral immune parameters. The calculated IBR values based on multi-level biomarkers revealed the size-dependent toxic differences of PS-NPs > PS-MPs. The relative expression levels of GDH and GS mRNA showed first rise and then fall trends after exposure to ammonia, and 100 nm PS-NPs had a more profound impact on suppressing ammonia detoxification compared with 20 µm PS-MPs. Moreover, the expression of Hsp90, Hsp70, CL, TLR, and CASP2 genes were all down-regulated by ammonia exposure. Taken together of IBR results, ammonia stress test and pathogen challenge, we deduced that dietary 100 nm PS-NPs are more potentially hazardous than 20 µm PS-MPs. These findings provide valuable information for understanding the size-dependent toxic effects of PS-N/MPs and early risk warning on marine invertebrates.

Transcriptomic analyses of the acute aerial and ammonia stress response in the gill and liver of large-scale loach (Paramisgurnus dabryanus).

The large-scale loach (Paramisgurnus dabryanus) is one of the most commercially important cultured species. Ammonia nitrogen accumulation is one of the key issue which limited production and animal health in aquaculture, but few of information is available on the molecular mechanisms of ammonia detoxification. We performed transcriptomic analyses of the gill and liver of large-scale loach subjected to 48 h of aerial and ammonia exposure. We obtained 47,473,424 to 56,791,496 clean reads from the aerial exposure, ammonia exposure and control groups, assembled and clustered a total of 92,658 unigenes with an average length of 909 bp and N50 of 1787 bp. Totals of 489/145 and 424/140 differentially expressed genes (DEGs) were detected in gill/liver of large-scale loach after aerial and ammonia exposure through comparative transcriptome analyses, respectively. In addition, totals of 43 gene ontology (GO) terms and 266 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified. After aerial and ammonia exposure, amino acid metabolism pathways in liver of large-scale loach were significantly enriched, suggesting that large-scale loach responded to high exogenous and endogenous ammonia stress by enhancing amino acid metabolism. Besides, the expression of several ammonia transporters (i.e., Rhesus glycoproteins and Aquaporins) in gill of large-scale loach were markedly changed after 48 h of aerial exposure, suggesting that large-scale loach responded to high endogenous ammonia stress by regulating the expression of Rh glycoproteins and Aqps related genes in gill. The results provide valuable information on the molecular mechanism of ammonia detoxification of large-scale loach to endogenous and environmental ammonia loading, will facilitate the molecular assisted breeding of ammonia resistant varieties, and will offer beneficial efforts for establishing an environmental-friendly and sustainable aquaculture industry.

The role of intestinal bacteria in the ammonia detoxification ability of teleost fish.

Protein catabolism during digestion generates appreciable levels of ammonia in the gastrointestinal tract (GIT) lumen. Amelioration by the enterocyte, via enzymes such as glutamine synthetase (GS), glutamate dehydrogenase (GDH), and alanine and aspartate aminotransferases (ALT; AST), is found in teleost fish. Conservation of these enzymes across bacterial phyla suggests that the GIT microbiome could also contribute to ammonia detoxification by providing supplemental activity. Hence, the GIT microbiome, enzyme activities and ammonia detoxification were investigated in two fish occupying dissimilar niches: the carnivorous rainbow darter and the algivorous central stoneroller. There was a strong effect of fish species on the activity levels of GS, GDH, AST and ALT, as well as GIT lumen ammonia concentration, and bacterial composition of the GIT microbiome. Furthermore, removal of the intestinal bacteria impacted intestinal activities of GS and ALT in the herbivorous fish but not in the carnivore. The repeatability and robustness of this relationship was tested across field locations and years. Within an individual waterbody, there was no impact of sampling location on any of these factors. However, different waterbodies affected enzyme activities and luminal ammonia concentrations in both fish, while only the central stoneroller intestinal bacteria populations varied. Overall, a relationship between GIT bacteria, enzyme activity and ammonia detoxification was observed in herbivorous fish while the carnivorous fish displayed a correlation between enzyme activity and ammonia detoxification alone that was independent of the GIT microbiome. This could suggest that carnivorous fish are less dependent on non-host mechanisms for ammonia regulation in the GIT.

Experimental bio-artificial liver: Importance of the architectural design on ammonia detoxification performance.

AIM: To determine the influence of the construction design over the biological component's performance in an experimental bio-artificial liver (BAL) device. METHODS: Two BAL models for liver microorgans (LMOs) were constructed. First, we constructed a cylindrical BAL and tested it without the biological component to establish its correct functioning. Samples of blood and biological compartment (BC) fluid were taken after 0, 60, and 120 min of perfusion. Osmolality, hematocrit, ammonia and glucose concentrations, lactate dehydrogenase (LDH) release (as a LMO viability parameter), and oxygen consumption and ammonia metabolizing capacity (as LMO functionality parameters) were determined. CPSI and OTC gene expression and function were measured. The second BAL, a "flat bottom" model, was constructed using a 25 cm2 culture flask while maintaining all other components between the models. The BC of both BALs had the same capacity (approximately 50 cm3) and both were manipulated with the same perfusion system. The performances of the two BALs were compared to show the influence of architecture. RESULTS: The cylindrical BAL showed a good exchange of fluids and metabolites between blood and the BC, reflected by the matching of osmolalities, and glucose and ammonia concentration ratios after 120 min of perfusion. No hemoconcentration was detected, the hematocrit levels remained stable during the whole study, and the minimal percentage of hemolysis (0.65% ± 0.10%) observed was due to the action of the peristaltic pump. When LMOs were used as biological component of this BAL they showed similar values to the ones obtained in a Normothermic Reoxygenation System (NRS) for almost all the parameters assayed. After 120 min, the results obtained were: LDH release (%): 14.7 ± 3.1 in the BAL and 15.5 ± 3.2 in the NRS (n = 6); oxygen consumption (µmol/min·g wet tissue): 1.16 ± 0.21 in the BAL and 0.84 ± 0.15 in the NRS (n = 6); relative expression of Cps1 and Otc: 0.63 ± 0.12 and 0.67 ± 0.20, respectively, in the BAL, and 0.86 ± 0.10 and 0.82 ± 0.07, respectively, in the NRS (n = 3); enzymatic activity of CPSI and OTC (U/g wet tissue): 3.03 ± 0.86 and 222.0 ± 23.5, respectively, in the BAL, and 3.12 ± 0.73 and 228.8 ± 32.8, respectively, in the NRS (n = 3). In spite of these similarities, LMOs as a biological component of the cylindrical BAL were not able to detoxify ammonia at a significant level (not detected vs 35.1% ± 7.0% of the initial 1 mM NH4 + dose in NRS, n = 6). Therefore, we built a second BAL with an entirely different design that offers a flat base BC. When LMOs were placed in this "flat bottom" device they were able to detoxify 49.3% ± 8.8% of the initial ammonia overload after 120 min of perfusion (n = 6), with a detoxification capacity of 13.2 ± 2.2 µmol/g wet tissue. CONCLUSION: In this work, we demonstrate the importance of adapting the BAL architecture to the biological component characteristics to obtain an adequate BAL performance.

Muscle wasting and branched-chain amino acid, alpha-ketoglutarate, and ATP depletion in a rat model of liver cirrhosis.

The aim of the study was to examine whether a rat model of liver cirrhosis induced by carbon tetrachloride (CCl4) is a suitable model of muscle wasting and alterations in amino acid metabolism in cirrhotic humans. Rats were treated by intragastric gavage of CCl4 or vehicle for 45 days. Blood plasma and different muscle types-tibialis anterior (mostly white fibres), soleus (red muscle) and extensor digitorum longus (white muscle) - were analysed at the end of the study. Characteristic biomarkers of impaired hepatic function were found in the plasma of cirrhotic animals. The weights and protein contents of all muscles of CCl4-treated animals were lower when compared with controls. Increased concentrations of glutamine (GLN) and aromatic amino acids (phenylalanine and tyrosine) and decreased concentrations of branched-chain amino acids (BCAA), glutamate (GLU), alanine and aspartate were found in plasma and muscles. In the soleus muscle, GLN increased more and GLU and BCAA decreased less than in the extensor digitorum and tibialis muscles. Increased chymotrypsin-like activity (indicating enhanced proteolysis) and decreased α-ketoglutarate and ATP levels were found in muscles of cirrhotic animals. ATP concentration also decreased in blood plasma. It is concluded that a rat model of CCl4-induced cirrhosis is a valid model for the investigation of hepatic cachexia that exhibits alterations in line with a theory of role of ammonia in pathogenesis of BCAA depletion, citric cycle and mitochondria dysfunction, and muscle wasting in cirrhotic subjects. The findings indicate more effective ammonia detoxification to GLN in red than in white muscles.

The activity of the carbamoyl phosphate synthase 1 promoter in human liver-derived cells is dependent on hepatocyte nuclear factor 3-beta.

Carbamoyl phosphate synthase 1 (CPS1) is the rate-limiting enzyme in the first step of the urea cycle and an indispensable enzyme in the metabolism of human liver. However, CPS1 epigenetic regulation involves promoter analysis and the role of liver-enriched transcription factors (LETFs), which is not fully elucidated. In this work, the promoter region of hCPS1 gene was cloned, and its activity was investigated. An LETF, hepatocyte nuclear factor 3-beta (HNF3ß), was found to promote the transcriptional expression of CPS1 in liver-derived cell lines. In addition, dual-luciferase reporter assay shows that the essential binding sites of the HNF3ß may exist in the oligonucleotide -70 nt to +73 nt. Two putative binding sites are available for HNF3ß. Mutation analysis results show that the binding site 2 of HNF3ß was effective, and the transcriptional activity of CPS1 promoter significantly decreased after mutation. Electrophoretic mobile shift assay (EMSA) and ChIP assay confirmed that HNF3ß can interact with the binding site in the CPS1 promoter region of -70 nt to +73 nt promoter region in vivo and in vitro to regulate the transcription of CPS1. Moreover, HNF3ß overexpression enhanced the transcription of CPS1 and consequently improved the mRNA and protein levels of CPS1, whereas the knockdown of HNF3ß showed the opposite effects. Finally, urea production in cells was measured, and ammonia detoxification improved significantly in cells after transfection with HNF3ß. HNF3ß plays a vital role in regulation of CPS1 gene and could promote the metabolism of ammonia by regulating CPS1 expression.

Evidence for Natural Selection in Nucleotide Content Relationships Based on Complete Mitochondrial Genomes: Strong Effect of Guanine Content on Separation between Terrestrial and Aquatic Vertebrates.

The complete vertebrate mitochondrial genome consists of 13 coding genes. We used this genome to investigate the existence of natural selection in vertebrate evolution. From the complete mitochondrial genomes, we predicted nucleotide contents and then separated these values into coding and non-coding regions. When nucleotide contents of a coding or non-coding region were plotted against the nucleotide content of the complete mitochondrial genomes, we obtained linear regression lines only between homonucleotides and their analogs. On every plot using G or A content purine, G content in aquatic vertebrates was higher than that in terrestrial vertebrates, while A content in aquatic vertebrates was lower than that in terrestrial vertebrates. Based on these relationships, vertebrates were separated into two groups, terrestrial and aquatic. However, using C or T content pyrimidine, clear separation between these two groups was not obtained. The hagfish (Eptatretus burgeri) was further separated from both terrestrial and aquatic vertebrates. Based on these results, nucleotide content relationships predicted from the complete vertebrate mitochondrial genomes reveal the existence of natural selection based on evolutionary separation between terrestrial and aquatic vertebrate groups. In addition, we propose that separation of the two groups might be linked to ammonia detoxification based on high G and low A contents, which encode Glu rich and Lys poor proteins.

How useful are clinical liver function tests in in vitro human hepatotoxicity assays?

In preclinical hepatotoxicity testing cell based assays are frequently employed. However, prediction of clinical drug induced liver injury (DILI) remains a major challenge. Here we examined the usefulness of frequently employed markers of hepatocellular injury in cultures of primary human hepatocytes (PHH) in response to treatment with either paracetamol, rifampicin, petadolex and/or amiodarone. The changes in the metabolic competency (urea and albumin) and cellular injury (AST, ALT, ALP, LDH, γGT and succinate dehydrogenase) were determined at therapeutic and above drug concentrations as to evaluate the utility of these markers in in vitro systems. Initially, treatment of PHH with any of the drugs caused a statistically significant reduction in enzyme activities to suggest a switch from basic amino acid metabolism towards induced detoxification. However, treatment for prolonged periods of time caused cytolysis, as evidenced by the significant rise in extracellular LDH and the concomitant increase in ALT and AST activity. Notably, amongst the various endpoints studied, urea was best to demonstrate dose dependent metabolic stress, while other markers of hepatocellular injury were highly variable. Taken collectively, urea measurement proofed to be robust in predicting hepatocellular stress; therefore it should be included in preclinical testing strategies for an improved prediction of DILI.