Growth hormone and rat liver mitochondria effects on urea cycle enzymes

Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.     

Nutritional influences on the distribution of the urea cycle: intermediates in isolated hepatocytes

After the urea cycle was proposed, considerable efforts were put forth to identify critical intermediates. This was then followed by studies of dietary and nutritional control of urea cycle enzyme activity and allosteric effectors of urea cycle enzymes. Correlation of urea cycle enzyme activity with isolated cell experiments indicated conditions where enzyme activity would be rate limiting. At physiological levels of ammonia the activation of carbamoyl-phosphate synthetase (EC 6.3.4.16) by N-acetylglutamate (NAG) is important. Various levels of NAG corresponded well with changes in the rate of citrulline and urea synthesis. Arginine was found to be an allosteric activator of N-acetylglutamate synthetase (EC 2.3.1.1). Therefore, it was possible that the rate of carbamoyl phosphate synthesis was dependent on the level of urea cycle intermediates, particularly arginine. Evidence for arginine in the regulation of NAG synthesis is not as clear as for NAG on carbamoyl phosphate synthetase I. The concentration of hepatic arginine is not necessarily an indication of the mitochondrial concentration. Only mitochondrial arginine stimulates the N-acetylglutamate synthetase. Recent studies indicate that the mitochondrial concentration of arginine is higher than the cytosolic concentration and is well above the Ka for N-acetylglutamate synthetase. Therefore, it appears that changes in arginine concentration are not physiologically important in regulating levels of NAG. However, it is possible that responses to the effector may vary with time after eating, and it may be this responsiveness that controls the level of NAG and thereby urea synthesis.     

Mitochondrial turnover in liver is fast in vivo and is accelerated by dietary restriction: application of a simple dynamic model

'Mitochondrial dysfunction', which may result from an accumulation of damaged mitochondria in cells due to a slowed-down rate of mitochondrial turnover and inadequate removal of damaged mitochondria during aging, has been implicated as both cause and consequence of the aging process and a number of age-related pathologies. Despite growing interest in mitochondrial function during aging, published data on mitochondrial turnover are scarce, and differ from each other by up to one order of magnitude. Here we demonstrate that re-utilization of the radioactively labelled precursor in pulse-chase assays is the most likely cause of significant overestimation of mitochondrial turnover rates. We performed a classic radioactive label pulse-chase experiment using (14)C NaHCO(3), whose (14)C is incorporated into various amino acids, to measure mitochondrial turnover in mouse liver. In this system, the activity of the urea cycle greatly limited arginine dependent label re-utilization, but not that of other amino acids. We used information from tissues that do not have an active urea cycle (brain and muscle) to estimate the extent of label re-utilization with a dynamic mathematical model. We estimated the actual liver mitochondrial half life as only 1.83 days, and this decreased to 1.16 days following 3 months of dietary restriction, supporting the hypothesis that this intervention might promote mitochondrial turnover as a part of its beneficial effects.     

Urea transporter B downregulates polyamines levels in melanoma B16 cells via p53 activation

Urea transporter B (UT-B, encoded by the SLC14A1 gene) is a membrane channel protein involved in urea transmembrane transport. Compared with normal tissues, UT-B expression is significantly decreased in most tumours, especially melanoma. However, the UT-B role in tumorigenesis and development is still unclear. Herein, we investigated the effects of UT-B overexpression on polyamine metabolism and the urea cycle in murine melanoma B16 cells, to explore the roles of mitochondrial dysfunction and p53 activation in cell growth and polyamines metabolism. UT-B overexpression in B16 cells decreased cell growth, increased apoptosis, and significantly altered metabolic pathways related to the urea cycle, which were characterized by reduced production of urea and polyamines and increased production of nitric oxide. Subsequently, we observed that activation of the p53 pathway may be the main cause of the above phenomena. The p53 inhibitor pifithrin-α partially restored the production of polyamines, but the mitochondrial morphology and function were still impaired. Further treatment of UT-B-overexpressing B16 cells with reactive oxygen species scavenging agent N-acetyl-l-cysteine and coenzyme Q10 restored cell viability and mitochondrial function and increased polyamine production. In conclusion, UT-B overexpression caused mitochondrial dysfunction and increased oxidative stress in B16 cells, and then activated p53 expression, which may be one of the mechanisms leading to the decrease in intracellular polyamines.     

Mitochondrial urea cycle enzymes in rats treated with sodium benzoate

Since sodium benzoate, which is widely used to treat hyperammonemia its effect on mitochondrial urea cycle enzymes was investigated. its effect on mitochondrial urea cycle enzymes was investigated. Sodium benzoate was administered to urease treated hyperammonemic rats and controls. In both groups no interference with the activity of carbamylphosphate synthetase, ornithine carbamyltransferase and N-acetylglutamate synthetase in the liver could be observed at concentrations of benzoate in plasma found in hyperammonemic patients. Careful monitoring of plasma levels reduces benzoate toxicity as shown in a patient with argininosuccinic aciduria.     

Green tea polyphenols boost gut-microbiota-dependent mitochondrial TCA and urea cycles in Sprague–Dawley rats

Green tea polyphenols (GTPs) were found to boost mammal energy conversion by modulating gut-microbial community structure, gene orthologs and metabolic pathways. Here we examined the metabolites present in the gut-microbiota-dependent mitochondrial tricarboxylic acid (TCA) cycle and urea cycle using hydrophilic interaction liquid chromatography (HILIC)-heated electrospray ionization (HESI)-tandem liquid chromatogram mass spectrometry (LC–MS). Six groups (n=12) of Sprague–Dawley rats (6-mo, ~250 g) were administered with water containing 0%, 0.5%, and 1.5% GTPs (wt/vol or g/dL). Gut-content samples were collected at 3- and 6-mo. Untargeted metabolomics detected 2177 features, with 91 features demonstrating significant dose- and time-dependencies on the GTPs treatment. Targeted metabolomics analysis revealed remarkable changes of 39 metabolites in the mitochondrial TCA cycle and urea cycle, including argininosuccunic acid (0.9-fold vs control), dihydrouracil (1.14-fold vs control), fumaric acid (1.19-fold vs control), malic acid (2.17-fold vs control), citrulline (1.86-fold vs control), and succinic acid (0.4-fold vs control). The untargeted metabolomics data were mined using bioinformatics approaches, such as analysis of variance-simultaneous component analysis (ASCA), enrichment pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping analysis. The results of 16S rRNA survey, metagenomics analysis, and metabolomics analysis were extrapolated and integrated using databases of Integrated Microbial Genomes and Microbiomes (IMG/M) and KEGG. Our analysis demonstrates that GTPs enhance energy conversion by boosting mitochondrial TCA cycle and urea cycle of gut-microbiota in rats. This metabolic modulation is achieved by enriching many gene orthologs, following the increase of beneficial microbials in families C. Ruminococcaceae, C. Lachnospiraceae and B. Bacteroidaceae.     

Does fish represent an intermediate stage in the evolution of ureotelic cytosolic arginase I?

Arginase catalyses the last step of the urea cycle. At least two isoenzymes of arginase are known; cytosolic ARG I and mitochondrial ARG II. ARG I is predominantly expressed in liver cytosol, as a part of urea cycle in ureotelic animals. The second isoform ARG II is primarily responsible for non-ureogenic functions, expressed in mitochondria of both hepatic and non-hepatic tissues in most vertebrates. Most micro-organisms and invertebrates are known to have only one type of arginase, whose function is unrelated to ornithine-urea cycle (OUC). However, in ureo-osmotic marine elasmobranchs arginase is localized in liver mitochondria as a part of OUC to synthesize urea for osmoregulation. An evolutionary transition occurred in arginase enzyme in terrestrial ureotelic vertebrates, with the evolution of ARG I from a pre-existing ancestral mitochondrial ARG II. This cytosolic ARG I activity is supposed to have first appeared in lung fishes, but the 40% and 60% distribution of arginase I and II activity in liver and kidney tissue of Heteropneustes fossilis indicates reconsideration of the above fact.     

Regulation of argininosuccinate synthetase level by corticosteroid and pancreatic hormones during perinatal period

The urea cycle takes place in the hepatocyte of ureothelic animals. The conversion of ammonia into urea involves five reactions. The first 2 take place in the matrix of the mitochondria, the last 2 occur in the cytosol. Argininosuccinate synthetase (AS) is the third reaction of the urea cycle. It catalyses the condensation of citrulline and aspartate into arginonosuccinate. We have previously reported that rat AS activity was present in the cytosol and the outer membrane of the mitochondria. We have shown that, at the activity level, the colocation of AS was changing during fetal and neonatal development and was under the control of corticosteroid and pancreatic hormones. However, an unresolved issue was whether both AS had the same specific activity and that their location was changing during ontogenesis or that the specific activities of mitochondrial and cytosolic enzymes were different and/or modified during this period. In the present report, we compared the compartmentalization of AS activity and protein level in the fetus, the new-born and the adult rat and the role of corticosteroid and pancreatic hormones. Specific activities of both AS remained unchanged during ontogenesis. Glucocorticoids induced an increase in mitochondrial AS while glucagon appeared to induce a concomitant decrease in the level of mitochondrial AS and an increase in cytosolic AS.     

Hormonal induction of hepatic mitochondrial ornithine/citrulline transporter mRNA

The urea cycle, which involves enzymes located in both the mitochondrion and cytoplasm, requires transport of ornithine and citrulline across the mitochondrial membrane by the ornithine/citrulline antiporter ORNT1. Expression of the urea cycle enzymes can change dramatically in response to hormones, but it is not known whether ORNT1 expression also is hormonally regulated. This study therefore tested the hypothesis that ORNT1 mRNA levels in hepatocytes are induced by cAMP and glucocorticoid as are the urea cycle enzyme mRNAs. ORNT1 mRNA was rapidly induced by a cAMP analog and dexamethasone in cultured rat hepatocytes and there was a strong synergistic response to a combination of these agents. Ongoing protein synthesis was required for induction of ORNT1 mRNA by dexamethasone but not by cAMP, suggesting that the dexamethasone response required an accessory factor. Thus, hormonal regulation of ORNT1 mRNA in hepatocytes is coordinated with that of mRNAs encoding the urea cycle enzymes.     

Cell cycle related studies on thymidine kinase and its isoenzymes in Ehrlich ascites tumours

Thymidine kinase (TK) activity was measured in relation to the cell cycle of in vivo growing ascites tumour cells. The cells were synchronized by means of centrifugal elutriation and the cell cycle composition of the cell fractions was determined by flow cytometry. TK activity was low in G1, increased during S phase and declined in G2. A half-life of TK activity of about 45 min was found throughout the cell cycle. Four isoenzymes at pI values of 4.1, 5.3, 6.9 and 8.3, denoted as isoenzymes 1-4, were identified using isoelectric focusing. Isoenzymes 3 and 4 were responsible for the profound cell cycle related changes in the TK activity. Corresponding isoenzymes were also found in the fetal mouse liver. In the adult mouse liver isoenzyme 2 was the dominating isoenzyme. The half-life of the isoenzymes was in the same range as for the total TK activity. We conclude that the low TK activity in G1 is due to degradation of the enzyme in G2 at a normal rate combined with an arrest in the synthesis of TK. We also conclude that isoenzyme 4 and the intermediate isoenzyme 3, which had earlier been suggested to be a mitochondrial form of TK, in fact represent cytoplasmatic forms of TK. According to cell cycle and pI studies, isoenzyme 2 belongs to the mitochondrial form. Studies with various phosphor donors and specific substrates, however, indicate that it also contains a cytoplasmic component.     

Stabilization of mitochondrial and microsomal function by polysaccharide of Ulva lactuca on D-Galactosamine induced hepatitis in rats

In this study we used liver mitochondrial and microsomal fraction from rats pretreated with seaweed Ulva lactuca polysaccharide extract (ULP - 200 mg/kg body weight, daily for 21 days, oral gavage) on D-Galactosamine (500 mg/kg body weight, intraperitoneally) challenge. Effectiveness of ULP was determined based on functional status of trichloro acetic acid (TCA), urea cycle, and microsomal enzymes. The composition of sulfate polysaccharide content such as total sugars, sulfate and uronic acid were examined. In addition the fine ultra structural changes were examined using electron microscopy (EM). We observed significant (p < 0.001) mitochondrial and microsomal abnormalities during liver damage by D-Galactosamine, consequently altering enzymes of energy metabolism. Electron microscopy of D-Galactosamine intoxicated rat liver tissue revealed the swelling and loss of mitochondrial cristae. Conversely the rats pretreated with ULP against D-Galactosamine challenge prevented (p < 0.05) the significant abnormality of TCA, microsomal enzymes and severity of mitochondria as observed in EM study in rats injected with D-Galactosamine alone. However no effective prevention was observed in urea cycle enzymes among D-Galactosamine and treatment group rats. These results showed the effectiveness of ULP in stabilizing the functional status of mitochondrial and microsomal membrane which might be due to the presence of sulfated polysaccharide that could prevented the oxidative stress induced by D-Galactosamine intoxication.     

The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.     

Discovery and Confirmation of O-GlcNAcylated Proteins in Rat Liver Mitochondria by Combination of Mass Spectrometry and Immunological Methods

O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.     

Agmatine stimulates hepatic fatty acid oxidation: a possible mechanism for up-regulation of ureagenesis

We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled beta-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled beta-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial beta-oxidation and suggest a coupling between the stimulation of hepatic beta-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently.     

Phycocyanobilin synthesis in the unicellular rhodophyte Cyanidium caldarium.

A method for the determination of the half-life of mitochondrial translation products in yeast in vivo is proposed. The method uses inhibitors of cytoplasmic and mitochondrial protein synthesis and is based on double-labelling pulse-chase techniques, the second label being used to estimate 'post-incorporation' during the 'chase'. For the first time the difference between post-incroporation and the widely known recycling of the label is considered. These studies show that, in the turnover of mitochondrial translation products, the problem is of post-incorporation into mitochondria (especially from the cell sap) is predominant. The results obtained with this procedure indicate that the half-life of the products of mitochondrial protein synthesis in yeast at the late-exponential phase is about 60 min. The results suggest that mitochondrial transplantation products are subject to proteolysis to acid-soluble forms.     

Differential expression and glycative damage affect specific mitochondrial proteins with aging in rat liver

Aging is accompanied by the gradual deterioration of cell functions. Particularly, mitochondrial dysfunction, associated with an accumulation of damaged proteins, is of key importance due to the central role of these organelles in cellular metabolism. However, the detailed molecular mechanisms involved in such impairment have not been completely elucidated. In the present study, proteomic analyses looking at both changes at the expression level as well as to glycative modifications of the mitochondrial proteome were performed. Two-dimensional difference gel electrophoresis analysis revealed 16 differentially expressed proteins with aging. Thirteen exhibited a decreased expression and are crucial enzymes related to OXPHOS chain complex I/V components, TCA cycle or fatty acid β-oxidation reaction. On the other hand, 2 enzymes involved in fatty acid β-oxidation cycle were increased in aged mitochondria. Immunodetection and further identification of glycated proteins disclosed a set of advanced glycation endproduct-modified proteins, including 6 enzymes involved in the fatty acid β-oxidation process, and 2 enzymes of the TCA/urea cycles. A crucial antioxidant enzyme, catalase, was among the most strongly glycated proteins. In addition, several AGE-damaged enzymes (aldehyde dehydrogenase 2, medium chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA dehydrogenase) exhibited a decreased activity with age. Taken together, these data suggest that liver mitochondria in old rats suffer from a decline in their capacity for energy production, due to (i) decreased expression of OXPHOS complex I/V components and (ii) glycative damage to key fatty acid β-oxidation and TCA/urea cycle enzymes.     

A metabolomics approach to investigate the proceedings of mitochondrial dysfunction in rats from prediabetes to diabetes

BackgroundDiabetes mellitus (DM) is a leading cause of preventable cardiovascular disease, but the metabolic changes from prediabetes to diabetes have not been fully clarified. This study implemented a metabolomics profiling platform to investigate the variations of metabolites and to elucidate their global profiling from metabolic syndrome to DM. Methods: Male Sprague-Dawley rats (n = 44) were divided into four groups. Three groups were separately fed with a normal diet, a high-fructose diet (HF), or a high-fat (HL) diet while one group was treated with streptozotocin. The HF and HL diet were meant to induce insulin resistance, obesity, and dyslipidemia, which known to induce DM. Results: The most significant metabolic variations in the DM group’s urine samples were the reduced release of citric acid cycle intermediates, the increase in acylcarnitines, and the decrease in urea excretion, all of which indicated energy metabolism abnormalities and mitochondrial dysfunction. Overall, the metabolic analysis revealed tryptophan metabolic pathway variations in the prediabetic phase, even though the mitochondrial function remains unaffected. Conclusion: This study show that widespread methylations and impaired tryptophan metabolism occur in metabolic syndrome and are then followed by a decline in citric acid cycle intermediates, indicating mitochondrial dysfunction in diabetes.     

Channeling of urea cycle intermediates in situ in permeabilized hepatocytes

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.