Carbamoyl Phosphate Synthetase Activity Research Articles

L-Ornithine-l-Aspartate (LOLA) Normalizes Metabolic Parameters in Models of Steatosis, Insulin Resistance and Metabolic Syndrome.

l-Ornithine- l-aspartate (LOLA) reduces toxic ammonium (NH3) plasma levels in hepatic encephalopathy. NH3 detoxification/excretion is achieved by its incorporation into urea and glutamine via activation of carbamoyl phosphate synthetase 1 (CSP1) by l-ornithine and stimulation of arginase by l-aspartate. We aimed at identifying additional molecular targets of LOLA as a potential treatment option for non-alcoholic fatty liver disease (NAFLD). In primary hepatocytes from NAFLD patients, urea cycle enzymes CSP1 and ornithine transcarbamylase (OTC) increase, while the catabolism of branched-chain amino acids (BCAAs) decreases with disease severity. In contrast, LOLA increased the expression rates of the BCAA enzyme transcripts bcat2, bckdha, and bckdk. In untreated HepG2 hepatoblastoma cells and HepG2-based models of steatosis, insulin resistance, and metabolic syndrome (the latter for the first time established herein), LOLA reduced the release of NH3; beneficially modulated the expression of genes related to fatty acid import/transport (cd36, cpt1), synthesis (fasn, scd1, ACC1), and regulation (srbf1); reduced cellular ATP and acetyl-CoA; and favorably modulated the expression of master regulators/genes of energy balance/mitochondrial biogenesis (AMPK-α, pgc1α). Moreover, LOLA reconstituted the depolarized mitochondrial membrane potential, while retaining mitochondrial integrity and avoiding induction of superoxide production. Most effects were concentration-dependent at ≤40 mM LOLA. We demonstrate for l-ornithine-l-aspartate a broad range of reconstituting effects on metabolic carriers and targets of catabolism/energy metabolism impaired in NAFLD. These findings strongly advocate further investigations to establish LOLA as a safe, efficacious, and cost-effective basic medication for preventing and/or alleviating NAFLD.

Impact of Negative Feedbacks on De Novo Pyrimidines Biosynthesis in Escherichia coli.

Earlier studies aimed at investigating the metabolism of endogenous nucleoside triphosphates in synchronous cultures of E. coli cells revealed an auto-oscillatory mode of functioning of the pyrimidine and purine nucleotide biosynthesis system, which the authors associated with the dynamics of cell division. Theoretically, this system has an intrinsic oscillatory potential, since the dynamics of its functioning are controlled through feedback mechanisms. The question of whether the nucleotide biosynthesis system has its own oscillatory circuit is still open. To address this issue, an integral mathematical model of pyrimidine biosynthesis was developed, taking into account all experimentally verified negative feedback in the regulation of enzymatic reactions, the data of which were obtained under in vitro conditions. Analysis of the dynamic modes of the model functioning has shown that in the pyrimidine biosynthesis system, both the steady-state and oscillatory functioning modes can be realized under certain sets of kinetic parameters that fit in the physiological boundaries of the investigated metabolic system. It has been demonstrated that the occurrence of the oscillatory nature of metabolite synthesis depended on the ratio of two parameters: the Hill coefficient, hUMP1-the nonlinearity of the UMP effect on the activity of carbamoyl-phosphate synthetase, and the parameter r characterizing the contribution of the noncompetitive mechanism of UTP inhibition to the regulation of the enzymatic reaction of UMP phosphorylation. Thus, it has been theoretically shown that the E. coli pyrimidine biosynthesis system possesses its own oscillatory circuit whose oscillatory potential depends to a significant degree on the mechanism of regulation of UMP kinase activity.

Complexed Crystal Structure of the Dihydroorotase Domain of Human CAD Protein with the Anticancer Drug 5-Fluorouracil.

Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.

214-LB: Activating and Disrupting the Liver–Alpha-Cell Axis Respectively Enhances and Impairs Amino Acid Metabolism and Alpha-Cell Growth

Amino acids control alpha cell secretion and growth, while glucagon stimulates hepatic amino acid uptake and ureagenesis, forming the liver-alpha cell axis. Patients with nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes have increased levels of glucagon and amino acids pointing to a disrupted liver-alpha cell axis. Both glucagon agonism and antagonism are explored for diabetes and NAFLD therapy but their effects on the axis are unclear. Female C57Bl/6JRj mice were treated with a long-acting glucagon analogue (GCGA, NNC9204-0043, Novo Nordisk A/S) or PBS twice daily, or once weekly with a GCGR antibody (GCGR Ab, REGN1193, Regeneron) or control antibody (Ctl. Ab, REGN1945, Regeneron) . After four weeks, total plasma amino acids were decreased by 48% in GCGA mice (P=0.0006) and increased by 285% in GCGR Ab mice (P<0.0001) versus week 0. At week 4, 18 of 19 amino acids were reduced in GCGA mice and increased in GCGR Ab mice. Asparagine changed the most in both groups, with a mean fold change of 0.21 and 7.50 in GCGA and GCGR Ab mice, respectively. Plasma urea index ([urea]/[amino acids]) was increased by 36% in GCGA mice (P=0.055) and decreased by 61% in GCGR Ab mice (P=0.001) . Liver RNA-sequencing revealed opposite effects of GCGA and GCGR Ab on expression of amino acid transporters (e.g. Slc43a) and ureagenesis genes (e.g. Ass1) . Activity of carbamoyl phosphate synthetase 1 was increased by 27% in GCGA versus PBS mice (P=0.007) and decreased by 33% in GCGR Ab mice versus Ctl. Ab (P=0.002) . GCGA lowered pancreas weight and glucagon content by 15% (P=0.004) and 95% (P<0.0001) compared to PBS. Conversely, GCGR Ab mice had a 34% increase in pancreas weight versus Ctl. Ab (P=0.0009) . Thus, chronically increased GCGR signaling leads to hypoaminoacidemia and decreased alpha cell mass, while chronically decreased GCGR signaling leads to hyperaminoacidemia and increased alpha cell mass. These changes must be considered when developing glucagon-based therapeutics. Disclosure E. E. Christensen: None. J. J. Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. N. J. Wewer albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc., Speaker's Bureau; Merck & Co., Inc., Mercodia AB. K. D. Galsgaard: None. C. D. Johansen: None. S. Trammell: None. A. B. Bomholt: None. J. Hunt: None. T. Kruse: Employee; Novo Nordisk. J. F. Lau: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. T. Grevengoed: None. Funding Nicolai J. Wewer Albrechtsen is supported by NNF Excellence Emerging Investigator Grant ? Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude (1052-00003B) . Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001) .

Formate induces a metabolic switch in nucleotide and energy metabolism

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.

Pharmacokinetic and Pharmacodynamic Properties of L-Ornithine L-Aspartate (LOLA) in Hepatic Encephalopathy.

l-Ornithine l-aspartate (LOLA), a stable salt of l-ornithine and l-aspartate, readily dissociates into its constituent amino acids that are readily absorbed by active transport, distributed, and metabolized. l-ornithine serves as an intermediary in the urea cycle in periportal hepatocytes in the liver and as an activator of carbamoyl phosphate synthetase, and, like l-aspartate, by transamination to glutamate via glutamine synthetase in perivenous hepatocytes as well as by skeletal muscle and brain. By way of these metabolic pathways, both amino acids participate in reactions whereby the ammonia molecule is incorporated into urea and glutamine and it is the nature, cellular, and biological location of these pathways that underpins the application of LOLA as an effective ammonia-lowering strategy widely used for the management and treatment of hepatic encephalopathy. These metabolic pathways were elucidated based upon studies in experimental animals and were confirmed by studies in patients with severe liver diseases. More recent studies suggest that LOLA may have additional direct hepatoprotective properties. Moreover, its use may result in improvements in skeletal muscle function in cirrhosis.

Formate Induces a Metabolic Switch in Nucleotide and Energy Metabolism

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.

Adipocyte Xbp1s overexpression drives uridine production and reduces obesity

ObjectiveThe spliced transcription factor Xbp1 (Xbp1s), a transducer of the unfolded protein response (UPR), regulates lipolysis. Lipolysis is stimulated by fasting when uridine synthesis is also activated in adipocytes. MethodsHere we have examined the regulatory role Xbp1s in stimulation of uridine biosynthesis in adipocytes and triglyceride mobilization using inducible mouse models. ResultsXbp1s is a key molecule involved in adipocyte uridine biosynthesis and release by activation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD), the rate-limiting enzyme for UMP biosynthesis. Adipocyte Xbp1s overexpression drives energy mobilization and protects mice from obesity through activation of the pyrimidine biosynthesis pathway. ConclusionThese observations reveal that Xbp1s is a potent stimulator of uridine production in adipocytes, enhancing lipolysis and invoking a potential anti-obesity strategy through the induction of a futile biosynthetic cycle.

Advances in methods for characterization of hepatic urea cycle enzymatic activity in HepaRG cells using UPLC-MS/MS

Current methodologies for the assessment of urea cycle (UC) enzymatic activity are insufficient to accurately evaluate this pathway in biological specimens where lower UC is expected. Liver cell lines, including HepaRG, have been described to have limited nitrogen fixation through the UC, limiting their applicability as biocomponents for Bioartificial Livers (BAL). This work aims to develop novel and sensitive analytical solutions using Mass Spectrometry-based methodology to measure the activity of four UC enzymes in human liver and HepaRG cells. Activity of carbamoyl-phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL) and arginase (ARG I and II) was determined on homogenates from normal human liver and HepaRG cells cultured in monolayer or in the AMC-BAL. Enzyme products were determined by stable-isotope dilution UPLC-MS/MS. Activity of CPS I, OTC and ARG I/II enzymes in HepaRG monolayer cultures was considerably lower than in human control livers albeit an increase was achieved in HepaRG-BAL cultures. Improved analytical assays developed for the study of UC enzyme activity, contributed to gain understanding of UC function in the HepaRG cell line. The decreased activity of CPS I suggests that it may be a potential rate-limiting factor underlying the low UC activity in this cell line.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

Transcriptome analysis reveals global regulation in response to CO2 supplementation in oleaginous microalga Coccomyxa subellipsoidea C-169.

BackgroundMicroalgae are emerging as suitable feedstock for renewable biofuel production and providing a promising way to alleviate green house gas CO2. Characterizing the metabolic pathways involved in the biosynthesis of energy-rich compounds and their global regulation upon elevated CO2 is necessary to explore the mechanism underlying rapid growth and lipid accumulation, so as to realize the full potential of these organisms as energy resources.ResultsIn the present study, 2 and 5 % CO2 increased growth rate and lipid accumulation in autotrophically cultured green alga Coccomyxa subellipsoidea C-169. Overall biomass productivity as 222 mg L−1 day−1 and fatty acid content as 48.5 % dry cell weight were attained in 2 % CO2, suggesting C-169 as a great candidate for lipid production via CO2 supplementation. Transcriptomic analysis of 2 % against 0.04 % CO2-cultured C-169 unveiled the global regulation of important metabolic processes. Other than enhancing gene expression in the Calvin cycle, C-169 upregulated the expression of phosphoenolpyruvate carboxylase, pyruvate carboxylase and carbamoyl-phosphate synthetase II to enhance the anaplerotic carbon assimilation reactions upon elevated CO2. Upregulation of ferredoxin and ferredoxin–NADP+ reductase implied that plentiful energy captured through photosynthesis was transferred through ferredoxin to sustain rapid growth and lipid accumulation. Genes involved in the glycolysis, TCA cycle and oxidative phosphorylation were predominantly upregulated presumably to provide abundant intermediates and metabolic energy for anabolism. Coordinated upregulation of nitrogen acquisition and assimilation genes, together with activation of specific carbamoyl-phosphate synthetase and ornithine pathway genes, might help C-169 to maintain carbon/nitrogen balance upon elevated CO2. Significant downregulation of fatty acid degradation genes, as well as the upregulation of fatty acid synthesis genes at the later stage might contribute to the tremendous lipid accumulation.ConclusionGlobal and collaborative regulation was employed by C-169 to assimilate more carbon and maintain carbon/nitrogen balance upon elevated CO2, which provide abundant carbon skeleton and affluent metabolic energy to sustain rapid growth and lipid accumulation. Data here for the first time bring significant insights into the regulatory profile of metabolism and acclimation to elevated CO2 in C-169, which provide important information for future metabolic engineering in the development of sustainable microalgae-based biofuels.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0571-5) contains supplementary material, which is available to authorized users.

Expression and specific activities of carbamoyl phosphate synthetase 1 in chronic hypoxic rats

Background: Urea biosynthesis is a very important process in the liver which needs ATP, CO2 and functional mitochondria or aerobic condition. Liver can adapt to hypoxic condition, generally and locally. This study aimed to analyze the effect of chronic hypoxia on liver urea biosynthesis as indicated by the level and specific activity of mRNA of carbamoyl phosphate synthetase 1 (CPS1), a key enzyme in urea biosynthesis in hypoxic rats.Methods: 20 male Sprague-Dawley rats were placed in hypoxic chamber supplied by a mixture of 10% O2 and 90% N2. Five rats were sacrificed at 1, 3, 5, and 7 days after exposure. Liver homogenates were analyzed for HIF-1 (hypoxia inducible factor-1) by ELISA, CPS1 mRNA by real time RT-PCR and CPS1 enzymatic specific activities by Pierson method. Data were analyzed by ANOVA test and Pearson correlation.Results: The HIF-1 in liver increased significantly, as well as CPS1 mRNA and CPS1 enzymatic activities (p<0.05). There was a strong correlation (r=0.618; p<0.01) between the level of CPS1 mRNA and CPS1 enzymatic activities, moderate correlation between HIF-1 and CPS1 mRNA (r=0.419; p<0.05) but no correlation between HIF-1 and CPS1 enzymatic activities. The study indicated that urea biosynthesis in liver was affected by hypoxia and partially under HIF-1 regulation. The study also found increase of urea and NH3 biosynthesis related to proteolysis as indicated by the decrease of total body weight and liver weight.Conclusion: There was an increase in the expression and specific activities of CPS1 in urea biosynthesis as a result of increasing proteolysis in chronic hypoxic condition.

Nitrogen Absorbed from the Large Intestine Increases Whole-Body Nitrogen Retention in Pigs Fed a Diet Deficient in Dispensable Amino Acid Nitrogen1–3

Background: Nitrogen absorption from the large intestine is considered of limited value for supporting body protein synthesis in animals and humans, but it may be of benefit when the dietary supply of nitrogen for synthesis of dispensable amino acids (DAAs) is deficient.Objective: A whole-body nitrogen balance study was conducted to evaluate the impact of nitrogen absorption from the large intestine of pigs fed a diet deficient in DAA nitrogen.Methods: Nine cecally cannulated barrows were fed a cornstarch and casein-based diet with a high indispensable amino acid (IAA) nitrogen to total nitrogen ratio (IAA:TN; 0.75). Pigs were randomly assigned to saline or 1 of 2 urea nitrogen infusion rates into the cecum (low and high, 1.5 and 3.0 g/d, respectively) following a 3 × 3 Latin square design. At the high urea nitrogen infusion rate, IAA:TN was 0.55. At slaughter, liver samples were taken to measure activity of carbamoyl phosphate synthetase I (CPS-I), glutamate dehydrogenase (GDH), and Gln synthetase (Gln-S).Results: Whole-body nitrogen retention improved with urea infusion (4.86 ± 0.20 g/d, 6.40 ± 0.21 g/d, and 7.75 ± 0.19 g/d for saline and low and high infusion rates, respectively; P < 0.05), as well as body weight gain. The marginal efficiency of using nitrogen absorbed from the large intestine for improving nitrogen retention was not affected by urea nitrogen infusion rate (P > 0.10). Enzyme activity of CPS-I or Gln-S was not different between treatments (P > 0.10), but GDH showed a trend for a positive linear response with increasing urea nitrogen infusion rate (P = 0.06).Conclusion: These results indicate that urea nitrogen absorbed from the large intestine is efficiently used for increasing body protein deposition when feeding pigs a diet deficient in DAA nitrogen.

Influence of feeding increasing levels of dry or modified wet corn distillers’ grains plus solubles in whole corn grain-based finishing diets on hepatic and renal mass, and glutathione peroxidase and urea cycle enzyme activities in finishing cattle

Salim, H., Wood, K. M., Cant, J. P. and Swanson, K. C. 2015. Influence of feeding increasing levels of dry or modified wet corn distillers’ grains plus solubles in whole corn grain-based finishing diets on hepatic and renal mass, and glutathione peroxidase and urea cycle enzyme activities in finishing cattle. Can. J. Anim. Sci. 95: 407–415. Forty-two cross-bred steers (BW=357±5.8 kg) fed whole corn grain-based finishing diets were used in a completely randomized block (60, 120, or 180 d on feed) design (2×3 factorial arrangement of treatments plus control) to determine the effect of inclusion level [0 (control), 16.7, 33.3, and 50% of diet DM) and form (dry (DDGS) or modified wet (MWDGS)] of distillers’ grains plus solubles (DGS) on hepatic and renal glutathione peroxidase (GPx) and hepatic urea cycle enzyme activities. Kidney weight (g kg−1of BW) increased linearly (P=0.004) with increasing inclusion levels of DGS. There were no effects (P≥0.11) of dietary treatment on hepatic and renal GPx activity (U g−1, U mg−1of protein, and kU liver−1). Hepatic carbamoyl phosphate synthetase activity (kU liver−1and U kg−1of BW) tended to linearly increase (P=0.09 and P=0.10, respectively) with increasing inclusion level of DGS. Hepatic ornithine transcarbamoylase and argininosuccinate synthetase activity (kU liver−1and U kg−1of BW) increased linearly (P≤0.05) with increasing inclusion levels of DGS. These data indicate that steers adapt to feeding up to 50% DGS by increasing kidney mass and activity of urea cycle enzymes in liver to allow for clearance of excess nitrogen. Also, hepatic and renal GPx activity, as an indicator of Se status, is not affected when typical finishing diets are fed.

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis.

Physiological and biochemical strategies for withstanding emersion in two galaxiid fishes

The galaxiid fishes of the Southern hemisphere display variable tolerance to aerial exposure. Brown mudfish (Neochanna apoda), for example, pseudoaestivate, inhabiting moist soil for months at a time, whereas inanga (Galaxias maculatus) emerse under unfavourable water conditions, but only for periods of a few hours. This study sought to identify the physiological and biochemical strategies that determine emersion tolerance in these species. Nitrogenous waste excretion was measured before and after an experimental emersion period (14days for mudfish, 6h for inanga). Both species showed significantly elevated ammonia “washout” upon return to water, but no increase in plasma or muscle ammonia. Post-emersion urea levels were elevated in plasma and muscle in both fish, however the extent of the accumulation did not indicate significant de novo urea production. This was supported by the lack of carbamoyl phosphate synthetase activity in tissues. Consequently, mudfish metabolism was examined to determine whether changes in parameters such as oxygen consumption, carbon dioxide excretion, and/or altered metabolic costs (represented by the key ionoregulatory enzyme Na+, K+-ATPase; NKA) could explain emersion tolerance. Oxygen consumption rates, already very low in immersed mudfish, were largely maintained over the course of emersion. Carbon dioxide excretion decreased during emersion, and a small, but significant, decrease in NKA was noted. These data suggest that the extended emersion capacity of mudfish may result from a generally low metabolic rate that is maintained throughout aerial exposure via cutaneous gas exchange, and which limits the production of potentially toxic nitrogenous waste.

Altered Nitrogen Balance and Decreased Urea Excretion in Male Rats Fed Cafeteria Diet Are Related to Arginine Availability

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids. We analyzed whether reduced urea excretion was a consequence of higher NOx; (nitrite, nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion. There were no differences in plasma nitrate or nitrite. NOx and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithine when compared with controls, whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.

52. Hypothermic preservation of rat liver microorgans (lmos) in ViaSpan® and BG35 (bes-gluconate-peg35) solutions: Study of ammonia metabolism during rewarming to evaluate their possible use as biological component of a BAL system

Bioartificial livers (BAL) were designed as a bridge to maintain patients with liver failure until they recover or receive a liver transplant. In these devices, the patient’s blood or plasma is circulated extra-corporeally through a bioreactor that houses a metabolically active component, as isolated hepatic cells or cultured cells. In this work, we tested LMOs from rat liver. LMOs are tissue slices that retain the microarchitecture of the liver lobe and its physiological characteristics. The use of LMOs is also attractive because it obviates the stages of cell isolation and cultivation. So, our group has set up a technique to manually obtain LMOs and constructed a flat-based BAL model suitable to use fresh LMOs. Moreover, we have developed a novel preservation solution, BG35, with similar efficacy than ViaSpan ® to protect LMOs against cold preservation injury. This would allow having the biological component viable, functional and ready to be used when is needed for our BAL. Ammonia accumulates in the blood of patients with liver failure, causing metabolic and neurological disturbances, being crucial that any cell or tissue to be employed in a BAL can perform this task. Since the Urea Cycle is the major pathway of ammonia removal, the objective of this work was to study gene expression and activity of Carbamoyl Phosphate Synthetase I (CPSI) and Ornithine Transcarbamylase (OTC), two key enzymes, after LMOs preservation in BG35 and ViaSpan ® (0 °C, 48 h). We have assayed in vitro the LMOs ability to detoxify an overload of ammonia in order to evaluate them for their successive application into a BAL. LMOs were manually cut (338 ± 27 μm thickness, n = 25). 50 LMOs were stored at 0 °C in 60 mL of BG35 or ViaSpan ® . Freshly-cut LMOs were used as controls. After cold storage, LMOs were rewarmed (37 °C, Krebs Henseleit Rewarming solution, 120 min) in a Dubnoff metabolic shaker under 95%O 2 :5% CO 2 atmosphere. Samples were taken after 60 and 120 min. All groups showed a good maintenance of their viability. After 120 min, the amount of LDH released by LMOs cold preserved in BG35 (20.1 ± 5.6%) and ViaSpan ® (23.4 ± 5.5 %) was similar to Controls (16.0 ± 4.4 %, n = 3). The preservation conditions did not affect ammonia metabolism: after 2 h of rewarming, LMOs cold preserved in BG35 and ViaSpan ® could detoxify 29.3 ± 7.9% and 25.7 ± 4.9% of the initial overload, similarly than Controls (29.7 ± 10.7%, n = 6). Though there were differences in relative mRNA levels between Controls and the two preserved groups, the values of CPSI and OTC enzymatic activity were comparable in all studied groups. In summary, we have shown that our preservation conditions did not affect ammonia metabolism and cold preserved LMOs maintain the physiological and biochemical liver functions tested, which allows their use as biological component of a BAL system.

Daytime restricted feeding modifies 24 h rhythmicity and subcellular distribution of liver glucocorticoid receptor and the urea cycle in rat liver

The timing system in mammals is formed by a set of peripheral biological clocks coordinated by a light-entrainable pacemaker located in the suprachiasmatic nucleus. Daytime restricted feeding (DRF) modifies the circadian control and uncouples the light-dependent physiological rhythmicity, food access becoming the principal external time cue. In these conditions, an alternative biological clock is expressed, the food-entrainable oscillator (FEO). Glucocorticoid hormones are an important part of the humoral mechanisms in the daily synchronisation of the metabolic response of peripheral oscillators by the timing system. A peak of circulating corticosterone has been reported before food access in DRF protocols. In the present study we explored in the liver the 24h variations of: (1) the subcellular distribution of glucocorticoid receptor (GCR), (2) the activities of the corticosterone-forming and NADPH-generating enzymes (11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) and hexose-6-phosphate dehydrogenase (H6PDH)), and, (3) parameters related with the urea cycle (circulating urea and activities of carbamoyl phosphate synthetase and ornithine transcarbamylase) elicited by DRF. The results showed that DRF promoted an increase of more than two times of the hepatic GCR, but exclusively in the cytosolic compartment, since the GCR in the nuclear fraction showed a reduction. No changes were observed in the activities of 11β-HSD-1 and H6PDH, but the rhythmicity of all of the urea cycle-related parameters was modified. It is concluded that liver glucocorticoid signalling and the urea cycle are responsive to feeding-restricted schedules and could be part of the FEO.

The HepaRG cell line is suitable for bioartificial liver application

For bioartificial liver application, cells should meet the following minimal requirements: ammonia elimination, drug metabolism and blood protein synthesis. Here we explore the suitability of HepaRG cells, a human cell line reported to differentiate into hepatocyte clusters and surrounding biliary epithelial-like cells at high density and after exposure to dimethyl sulfoxide (DMSO). The effect of carbamoyl-glutamate (CG), an activator of urea cycle enzyme carbamoylphosphate synthetase (CPS) was studied additionally. The effects of DMSO and/or CG were assessed in presence of 15NH 4Cl on HepaRG cells in monolayer. We tested hepatocyte-specific functions at transcript and biochemical level, cell damage parameters and performed immunostainings. Ureagenesis, ammonia/galactose elimination and albumin, glutamine synthetase and CPS transcript levels were higher in −DMSO than +DMSO cultures, probably due to a higher cell content and/or cluster-neighbouring regions contributing to their functionality. DMSO treatment increased cytochrome P450 ( CYP) transcript levels and CYP3A4 activity, but also cell damage and repressed hepatic functionality in cluster-neighbouring regions. The levels of ammonia elimination, apolipoprotein A-1 production, and transcription of CYP3A4, CYP2B6 and albumin reached those of primary hepatocytes in either the + or −DMSO cultures. Preconditioning with CG increased conversion of 15NH 4Cl into 15N-urea 4-fold only in −DMSO cultures. Hence, HepaRG cells show high metabolic and synthetic functionality in the absence of DMSO, however, their drug metabolism is only high in the presence of DMSO. An unparalleled broad hepatic functionality, suitable for bioartificial liver application, can be accomplished by combining CG treated −DMSO cultures with +DMSO cultures.