Dataset Information

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

ABSTRACT: The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.

SUBMITTER: He Y

PROVIDER: S-EPMC2843202 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

He Youji Y Hakvoort Theodorus B M TBM Köhler S Eleonore SE Vermeulen Jacqueline L M JLM de Waart D Rudi DR de Theije Chiel C Ten Have Gabrie A M GAM van Eijk Hans M H HMH Kunne Cindy C Labruyere Wilhelmina T WT Houten Sander M SM Sokolovic Milka M Ruijter Jan M JM Deutz Nicolaas E P NEP Lamers Wouter H WH

The Journal of biological chemistry 20100111 13

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed th ...[more]

PMID: 20064933

Similar Datasets

Project description:NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine) in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown) glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine) is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS structural elements associated with glutamine-utilizing capabilities.

Project description:Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 μM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

Project description:BackgroundHepatic fibrosis is a common pathologic stage in chronic liver disease development, which might ultimately lead to liver cirrhosis. Accumulating evidence suggests that adipose-derived stromal cells (ADSCs)-based therapies show excellent therapeutic potential in liver injury disease owing to its superior properties, including tissue repair ability and immunomodulation effect. However, cell-based therapy still limits to several problems, such as engraftment efficiency and immunoreaction, which impede the ADSCs-based therapeutics development. So, ADSCs-derived extracellular vesicles (EVs), especially for exosomes (ADSC-EXO), emerge as a promise cell-free therapeutics to ameliorate liver fibrosis. The effect and underlying mechanisms of ADSC-EXO in liver fibrosis remains blurred.MethodsHepatic fibrosis murine model was established by intraperitoneal sequential injecting the diethylnitrosamine (DEN) for two weeks and then carbon tetrachloride (CCl4) for six weeks. Subsequently, hepatic fibrosis mice were administrated with ADSC-EXO (10 μg/g) or PBS through tail vein infusion for three times in two weeks. To evaluate the anti-fibrotic capacity of ADSC-EXO, we detected liver morphology by histopathological examination, ECM deposition by serology test and Sirius Red staining, profibrogenic markers by qRT-PCR assay. LX-2 cells treated with TGF-β (10 ng/ml) for 12 h were conducted for evaluating ADSC-EXO effect on activated hepatic stellate cells (HSCs). RNA-seq was performed for further analysis of the underlying regulatory mechanisms of ADSC-EXO in liver fibrosis.ResultsIn this study, we obtained isolated ADSCs, collected and separated ADSCs-derived exosomes. We found that ADSC-EXO treatment could efficiently ameliorate DEN/CCl4-induced hepatic fibrosis by improving mice liver function and lessening hepatic ECM deposition. Moreover, ADSC-EXO intervention could reverse profibrogenic phenotypes both in vivo and in vitro, including HSCs activation depressed and profibrogenic markers inhibition. Additionally, RNA-seq analysis further determined that decreased glutamine synthetase (Glul) of perivenous hepatocytes in hepatic fibrosis mice could be dramatically up-regulated by ADSC-EXO treatment; meanwhile, glutamine and ammonia metabolism-associated key enzyme OAT was up-regulated and GLS2 was down-regulated by ADSC-EXO treatment in mice liver. In addition, glutamine synthetase inhibitor would erase ADSC-EXO therapeutic effect on hepatic fibrosis.ConclusionsThese findings demonstrated that ADSC-derived exosomes could efficiently alleviate hepatic fibrosis by suppressing HSCs activation and remodeling glutamine and ammonia metabolism mediated by hepatocellular glutamine synthetase, which might be a novel and promising anti-fibrotic therapeutics for hepatic fibrosis disease.

Project description:Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4-5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61-71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.

Dataset Information

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

Publications

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets