Induction of the five urea-cycle enzymes by glucagon in cultured foetal rat hepatocytes

Husson, Annie; Buquet, Catherine; Vaillant, R.

doi:10.1111/j.1432-0436.1987.tb00171.x

Cited by 21 publications

(17 citation statements)

References 35 publications

(27 reference statements)

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“…It has been well described that glucagon is able to stimulate ureagenesis by up‐regulating the expression of urea cycle enzymes 34–36. Thus, we decided to test if glucagon was able to induce hepatocyte mtAQP8 expression in vivo in the rat.…”

Section: Resultsmentioning

confidence: 99%

Ammonia detoxification via ureagenesis in rat hepatocytes involves mitochondrial aquaporin-8 channels

et al. 2013

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Hepatocyte mitochondrial ammonia detoxification via ureagenesis is critical for the prevention of hyperammonemia and hepatic encephalopathy. Aquaporin-8 (AQP8) channels facilitate the membrane transport of ammonia. Because AQP8 is expressed in hepatocyte inner mitochondrial membranes (IMMs), we studied whether mitochondrial AQP8 (mtAQP8) plays a role in ureagenesis from ammonia. Primary cultured rat hepatocytes were transfected with small interfering RNAs (siRNAs) targeting two different regions of the rat AQP8 molecule or with scrambled control siRNA. After 48 hours, the levels of mtAQP8 protein decreased by approximately 80% (P < 0.05) without affecting cell viability. mtAQP8 knockdown cells in the presence of ammonium chloride showed a decrease in ureagenesis of approximately 30% (P < 0.05). Glucagon strongly stimulated ureagenesis in control hepatocytes (1120%, P < 0.05) but induced no significant stimulation in mtAQP8 knockdown cells. Contrarily, mtAQP8 silencing induced no significant change in basal and glucagon-induced ureagenesis when glutamine or alanine was used as a source of nitrogen. Nuclear magnetic resonance studies using 15N-labeled ammonia confirmed that glucagon-induced 15N-labeled urea synthesis was markedly reduced in mtAQP8 knockdown hepatocytes (290%, P < 0.05). In vivo studies in rats showed that under glucagoninduced ureagenesis, hepatic mtAQP8 protein expression was markedly up-regulated (1160%, P < 0.05). Moreover, transport studies in liver IMM vesicles showed that glucagon increased the diffusional permeability to the ammonia analog [ 14 C]methylamine (180%, P < 0.05). Conclusion: Hepatocyte mtAQP8 channels facilitate the mitochondrial uptake of ammonia and its metabolism into urea, mainly under glucagon stimulation. This mechanism may be relevant to hepatic ammonia detoxification and in turn, avoid the deleterious effects of hyperammonemia. (HEPATOLOGY 2013;57:2061-2071 B y means of the urea cycle, hepatocytes metabolize waste nitrogen and prevent the deleterious consequences of hyperammonemia and hepatic encephalopathy. The initial and rate-controlling step in the urea cycle is the mitochondrial synthesis of carbamoyl-phosphate from ammonia by carbamyl phosphate synthetase 1 (CPS1), 1 a process that involves the mitochondrial uptake of ammonia and the metabolization of ammonia from amino acids such as glutamine and alanine. 2 However, the mechanisms involved in the mitochondrial entry of ammonia are still unknown (Fig. 1). Aquaporin-8 (AQP8) is a member of a family of homologous membrane channel proteins that facilitates the movement of water and certain small solutes across biological membranes. 3 Hepatocyte AQP8 is localized as a glycosylated 34-kDa protein in the canalicular plasma membrane domain and intracellular vesicles. 4-7 Canalicular AQP8 works as a water channel facilitating the movement of water molecules coupled to osmotic gradients, 8,9 a process that may play a role in hepatocyte bile formation and cholestasis. 7,10-13 A nonglycosylated 28-kDa form of the AQP8 protei...

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Section: Resultsmentioning

confidence: 99%

Ammonia detoxification via ureagenesis in rat hepatocytes involves mitochondrial aquaporin-8 channels

et al. 2013

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“…Opticell PICM-19 cultures (flight; 1FB, 4FB, and ground; 1GA, 4GB) were treated in situ by refeeding with glutamine-free Williams-E medium supplemented with 10% FBS, 1 mM ornithine, glucagon (100 ng/ml), 2-mercaptothanol (0.1 mM), HEPES (25 mM), and antibiotics for 48 h to stimulate urea production (Triebwasser and Freedland 1977;Metoki and Hommes 1984;Husson et al 1987). One-quarter pieces of the PICM-19 monolayers were then exposed to the same base medium with or without the addition of 2 mM ammonium chloride in 6-well plates for 24 h. Medium was collected, centrifuged at 2,000×g to remove cellular debris, and frozen at −80°C prior to analysis.…”

Section: Methodsmentioning

confidence: 99%

The effects of space flight and microgravity on the growth and differentiation of PICM-19 pig liver stem cells

Talbot

Caperna

Blomberg

et al. 2010

In Vitro Cell.Dev.Biol.-Animal

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The PICM-19 pig liver stem cell line was cultured in space for nearly 16 d on the STS-126 mission to assess the effects of spaceflight on the liver's parenchymal cells-PICM-19 cells to differentiate into either monolayers of fetal hepatocytes or 3-dimensional bile ductules (cholangiocytes). Semi-quantitative data included light microscopic assessments of final cell density, cell morphology, and response to glucagon stimulation and electron microscopic assessment of the cells' ultrastructural features and cell-to-cell connections and physical relationships. Quantitative assessments included assays of hepatocyte detoxification functions, i.e., inducible P450 activities and urea production and quantitation of the mRNA levels of several liver-related genes. Three post-passage age groups were included: 4-d-, 10-d-, and 14-d-old cultures. In comparing flight vs. ground-control cultures 17 h after the space shuttle's return to earth, no differences were found between the cultures with the exception being that some genes were differentially expressed. By light microscopy both young and older cultures, flight and ground, had grown and differentiated normally in the Opticell culture vessels. The PICM-19 cells had grown to approximately 75% confluency, had few signs of apoptosis or necrosis, and had either differentiated into monolayer patches of hepatocytes with biliary canaliculi visible between the cells or into 3-dimensional bile ductules with well-defined lumens. Ultrastructural features between flight and ground were similar with the PICM-19 cells displaying numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies, and occasional lipid vacuoles. Cell-to-cell arrangements were typical in both flight and ground-control samples; biliary canaliculi were well-formed between the PICM-19 cells, and the cells were sandwiched between the STO feeder cells. PICM-19 cells displayed inducible P450 activities. They produced urea in a glutamine-free medium and produced more urea in response to ammonia. The experiment's aim to gather preliminary data on the PICM-19 cell line's suitability as an in vitro model for assessments of liver function in microgravity was demonstrated, and differences between flight and ground-control cultures were minor.

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“…To test this possibility, puromycin (or cycloheximide) was added together with dexamethasone to 18.5-day hepatocytes and left for 24 h. Modulation of dexamethasone effect by insulin and glucagon. It was previously shown that the physiological variations of plasma insulin and glucagon concentrations during the perinatal period could control the development of the ASS activity [17,31,321. To specify the effects of these hormones at the mRNA level, 18.5-day hepatocytes were cultured with lo-' M insulin or glucagon, in the presence or absence of dexamethasone for 24 h. As illustrated by the autoradiograms of Fig.…”

Section: -Actinmentioning

confidence: 99%

Regulation of Argininosuccinate Synthetase mRNA Level in Rat Foetal Hepatocytes

Bourgeois

Harlin

Renouf

et al. 1997

European Journal of Biochemistry

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Expression of the hepatic gene for argininosuccinate synthase (ASS), one of the key enzymes of the urea cycle, was analysed during the perinatal period in the rat. To this end, the amount of specific mRNA was measured in the liver at various stages of development and in cultured foetal hepatocytes maintained in different hormonal conditions. The ASS mRNA was first detected in 15.5-day foetuses and its level increased concomitantly with a rise in the enzyme activity, suggesting that the appearance of the ASS activity reflects the turning on of specific gene transcription. This was demonstrated by run-on assay which showed an enhanced rate of transcription of the ASS gene during the perinatal period. When foetal hepatocytes were cultured with dexamethasone, a dose-dependent increase in ASS mRNA was measured, which was completely abolished by actinomycin D addition. The transcription rate of the gene was increased about twofold in the presence of the steroid, as measured by nuclear run-on assay. This transcriptional action could additionally require a protein factor since it could be inhibited by the simultaneous addition of puromycin. Insulin or glucagon respectively repressed or enhanced the dexamethasone-induced accumulation of ASS mRNA when added simultaneously with the steroid for 24 h. This developmental regulation of the ASS mRNA by glucocorticoids, insulin and glucagon could account for the modulation of the enzyme activity previously observed in vivo and in vitro in the foetal liver.Keywords: argininosuccinate synthetase; mRNA ; gene transcription ; cultured foetal hepatocyte; glucocorticoid.Argininosuccinate synthetase (ASS) catalyses the synthesis of argininosuccinate from aspartate and citrulline, and is one of the key enzymes of the urea cycle in Mammals [l]. This enzyme is expressed in many tissues, with highest expression in the liver and in the kidney, where it plays a role in the arginine synthesis [2], and in the testis [3].Regulation of expression of ASS in the adult rat liver appears complex since increased protein content in the diet, prolonged starvation [4] and administration of glucocorticoids or glucagon have been shown to increase its activity both in vivo [5, 61 and in vitro [7, 81. Most of these treatments also produced an accumulation of specific mRNA [9-111. The expression of this gene was shown to be also regulated by amino acids since it was stimulated by glutamine addition in cultured rat hepatocytes [12] and repressed by arginine in cultured human cellsIn the late foetal period, the urea cycle enzymes are actively synthesized in the liver and this differentiation largely depends on the foetal endocrine state [14-161. Previous studies have shown that the ASS activity appeared in the foetal rat liver during the last days of gestation and that its development depended on the presence of foetal adrenals [14, 151 and on plasma levels of glucagon and insulin [17]. In particular ASS activity did not ~3 1 . The aim of the present work was to characterize the expression of the gene of ASS ...

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Induction of the five urea-cycle enzymes by glucagon in cultured foetal rat hepatocytes

Cited by 21 publications

References 35 publications

Ammonia detoxification via ureagenesis in rat hepatocytes involves mitochondrial aquaporin-8 channels

Ammonia detoxification via ureagenesis in rat hepatocytes involves mitochondrial aquaporin-8 channels

The effects of space flight and microgravity on the growth and differentiation of PICM-19 pig liver stem cells

Regulation of Argininosuccinate Synthetase mRNA Level in Rat Foetal Hepatocytes

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