Hepatic glutaminase mRNA is confined to part of the urea cycle domain in the adult rodent liver lobule

Moorman, Antoon F.M.; Boer, P. A. J. De; Watford, Malcolm; Dingemanse, Maria A.; Lamers, Wouter H.

doi:10.1016/0014-5793(94)01230-x

Cited by 32 publications

(23 citation statements)

References 30 publications

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“…In situ hybridization on 4% formaldehyde-fixed livers was performed as described previously (43,65). Serial sections 7 m thick were probed for CPS and glutamine synthetase (GS) with the respective 35 S-labeled cRNA probes (6,26).…”

Section: Methodsmentioning

confidence: 99%

Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

Christoffels

Grange

Kaestner

et al. 1998

Molecular and Cellular Biology

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A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid-and cyclic AMPinducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor-and C/EBP␣-deficient mice, whereas the CPS mRNA was normally expressed in C/EBP␤ knockout mice and in HNF3␣ and -␥ double-knockout mice. (The role of HNF␤ could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.

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Section: Methodsmentioning

confidence: 99%

Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

Christoffels

Grange

Kaestner

et al. 1998

Molecular and Cellular Biology

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“…(a) Our primary research interest involves the analysis of gene expression in derivatives of the embryonic foregut (Moorman et al 1990(Moorman et al ,1994Rings et al 1992). (b) The tissue processing requires no specific protocol, and many similar serial sections can be made easily.…”

Section: Methodsmentioning

confidence: 99%

Towards Quantitative In Situ Hybridization

Jonker

Boer

Hoff

et al. 1997

J Histochem Cytochem.

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SUMMARYIn situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35 S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPSexpressing cells in total intestinal tissue.

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“…(41) In these mice, hepatic and plasma glutamine concentrations increase 12-fold and 25-fold, respectively, whereas suppression of the pericentral enzymic phenotype by hepatocyte-specific deletion of b-catenin has no such effect. (42) Accordingly, periportal hepatocytes are well equipped to take up and metabolize glutamine (4,27) because they express the glutamine transporter SLC38A5, (35) glutaminase-2 (EC 3.5.1.2), (43,44) and all enzymes of the urea cycle. (45,46) The trans-membranous Na 1 gradient mediates the uptake of >70% of plasma glutamine and causes it to accumulate intracellularly to 3-5 mmol/ kg.…”

Section: Periportal Hepatocytes Catabolize Glutaminementioning

confidence: 99%

Pivotal role of glutamine synthetase in ammonia detoxification

et al. 2016

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Glutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. Glutamine serves, with alanine, as a major nontoxic interorgan ammonia carrier. Elimination of hepatic GS expression in mice causes only mild hyperammonemia and hypoglutaminemia but a pronounced decrease in the whole-body muscle-to-fat ratio with increased myostatin expression in muscle. Using GS-knockout/liver and control mice and stepwise increments of enterally infused ammonia, we show that 35% of this ammonia is detoxified by hepatic GS and 35% by urea-cycle enzymes, while 30% is not cleared by the liver, independent of portal ammonia concentrations £ 2 mmol/L. Using both genetic (GSknockout/liver and GS-knockout/muscle) and pharmacological (methionine sulfoximine and dexamethasone) approaches to modulate GS activity, we further show that detoxification of stepwise increments of intravenously (jugular vein) infused ammonia is almost totally dependent on GS activity. Maximal ammonia-detoxifying capacity through either the enteral or the intravenous route is 160 lmol/hour in control mice. Using stable isotopes, we show that disposal of glutaminebound ammonia to urea (through mitochondrial glutaminase and carbamoylphosphate synthetase) depends on the rate of glutamine synthesis and increases from 7% in methionine sulfoximine-treated mice to 500% in dexamethasone-treated mice (control mice, 100%), without difference in total urea synthesis. Conclusions: Hepatic GS contributes to both enteral and systemic ammonia detoxification. Glutamine synthesis in the periphery (including that in pericentral hepatocytes) and glutamine catabolism in (periportal) hepatocytes represents the high-affinity ammonia-detoxifying system of the body. The dependence of glutamine-bound ammonia disposal to urea on the rate of glutamine synthesis suggests that enhancing peripheral glutamine synthesis is a promising strategy to treat hyperammonemia. Because total urea synthesis does not depend on glutamine synthesis, we hypothesize that glutamate dehydrogenase complements mitochondrial ammonia production. (HEPATOLOGY 2017;65:281-293). G lutamine synthetase (GS) catalyzes condensation of ammonia with glutamate to glutamine. (Note: NH 3 is protonated for 98% to NH 4 1 at physiological pH. We use the term "ammonia" to refer to the sum of NH 3 and NH 4 1 unless specified as either "NH 3 " or "NH 41 .") Glutamine serves, with alanine, as a major nontoxic interorgan ammonia shuttle in the body (1) and as an aminomoiety donor for the synthesis of nucleotides, amino acids, amino-sugars, and oxidized nicotinamide adenine dinucleotide. Its intracellular turnover rate exceeds that of all other amino acids. GS deficiency causes only moderate hyperammonemia, but affected humans and mice suffer from encephalopathy and die neonatally. (2,3) GS is predominantly expressed in the nervous system, kidney, and liver, that is, in established glutamine-consuming organs, (4)(5)(6) whereas skeletal muscle, with a much lower expression but large mass, is considered the main net ...

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Hepatic glutaminase mRNA is confined to part of the urea cycle domain in the adult rodent liver lobule

Cited by 32 publications

References 30 publications

Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

Towards Quantitative In Situ Hybridization

Pivotal role of glutamine synthetase in ammonia detoxification

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