The effects L-leucine on the synthesis of urea, glutamate and glutamine by isolated rat liver cells.
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ABSTRACT:
SUBMITTER: Mourao JM
PROVIDER: S-EPMC1165324 | biostudies-other | 1975 Feb
REPOSITORIES: biostudies-other
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Project description:Addition of phenylephrine to isolated perfused rat liver is followed by an increased 14CO2 production from [1-14C]glutamate, [1-14C]glutamine, [U-14C]proline and [3-14C]pyruvate, but by a decreased 14CO2 production from [1-14C]pyruvate. Simultaneously, there is a considerable decrease in tissue content of 2-oxoglutarate, glutamate and citrate. Stimulation of 14CO2 production from [1-14C]glutamate is also observed in the presence of amino-oxyacetate, suggesting a stimulation of glutamate dehydrogenase and 2-oxoglutarate dehydrogenase fluxes by phenylephrine. Inhibition of pyruvate dehydrogenase flux by phenylephrine is due to an increased 2-oxoglutarate dehydroxygenase flux. Phenylephrine stimulates glutaminase flux and inhibits glutamine synthetase flux to a similar extent, resulting in an increased hepatic glutamine uptake. Whereas the effects of NH4+ ions and phenylephrine on glutaminase flux were additive, activation of glutaminase by glucagon was considerably diminished in the presence of phenylephrine. The reported effects are largely overcome by prazosin, indicating the involvement of alpha-adrenergic receptors in the action of phenylephrine. It is concluded that stimulation of gluconeogenesis from various amino acids by phenylephrine is due to an increased flux through glutamate dehydrogenase and the citric acid cycle.
| S-EPMC1144094 | biostudies-other
Project description:The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver (P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4 (SIRT4) gene expression (P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1 (mTORC1) signaling (P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1 (S6K1) and ribosomal protein S6 (S6). Interestingly, the leucine-induced mTORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4. Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes (P > 0.05), while mTORC1 signaling was activated. Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes (P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating mTORC1/SIRT4 pathway in the liver of pigs.
| S-EPMC6116330 | biostudies-literature
Project description:In a non-recirculating system of isolated liver perfusion, stimulation of urea synthesis by NH4Cl is followed by a decrease of effluent pH by up to 0.2 pH unit. This effect is not observed when urea synthesis is inhibited by amino-oxyacetate or norvaline. When the urea formed by the liver is immediately hydrolysed with urease before the effluent perfusate reaches the pH electrode, the urea-synthesis-induced acidification is no longer observed. This indicates that accompanying alterations in hepatic metabolism after stimulation of urea synthesis, such as increased energy provision and consumption, are not responsible for the extracellular acidification, but that the effect is due to the formation of urea itself. The acidification of the extracellular space after stimulation of urea synthesis by NH4Cl is quantitatively explained by the consumption of 2 mol of HCO3-/mol of urea formed: 1 mol being incorporated into urea, the other being protonated to yield CO2 and H2O. The data match the theoretically predicted HCO3- consumption during ureogenesis and underline the role of hepatic urea synthesis for disposal of HCO3- by converting it into the excretable products CO2 and urea.
| S-EPMC1146814 | biostudies-other
Project description:1. On perfusion of livers from fed rats with a semi-synthetic medium containing no added amino acids there is a rapid release of glutamine during the first 15min (15.6+/-0.8mumol/h per g wet wt.; mean+/-s.e.m. of 35 experiments), followed by a low linear rate of production (3.6+/-0.3mumol/h per g wet wt.; mean+/-s.e.m. of three experiments). The rapid initial release can be accounted for as wash-out of preexisting intracellular glutamine. 2. Addition of readily permeating substrates, or substrate combinations, giving rise to intracellular glutamate or ammonia, or both, did not appreciably increase the rate of glutamine production over the endogenous rate. The maximum rate obtained was from proline plus alanine; even then the rate represented less than one-fortieth of the capacity of glutamine synthetase measured in vitro. 3. Complete inhibition of respiration in the perfusions [no erythrocytes in the medium; 1mm-cyanide; N(2)+CO(2) (95:5) in the gas phase] or perfusion with glutamine synthetase inhibitors [l-methionine dl-sulphoximine; dl-(+)-allo-delta-hydroxylysine] abolishes the low linear rate of glutamine synthesis, but not the initial rapid release of glutamine. 4. In livers from 48h-starved rats initial release (0-15min) of glutamine was decreased (10.6+/-1.1mumol/h per g wet wt.; mean+/-s.e.m. of 11 experiments) and the subsequent rate of glutamine production was lower than in livers from fed rats. Again proline plus alanine was the only substrate combination giving an increase significantly above the endogenous rate. 5. The rate of glutamine synthesis de novo by the liver is apparently unrelated to the tissue content of glutamate or ammonia. 6. The blood glutamine concentration is increased by 50% within 1h of elimination of the liver from the circulation of rats in vivo. 7. There is an output of glutamine by the brain under normal conditions; the mean arterio-venous difference for six rats was 0.023mumol/ml. 8. The high potential activity of liver glutamine synthetase appears to be inhibited by some unknown mechanism: the function of the liver under normal conditions is probably the disposal of glutamine produced by extrahepatic tissues.
| S-EPMC1177235 | biostudies-other
Project description:Glucagon injected into rats caused an increase in liver N-acetylglutamate content, and coincidentally the glutamate content decreased. The liver glutamine content decreased only after 10 min, consistent with an activation of glutaminase after a lag. These observations indicate that the increased N-acetylglutamate content was not due to an increase in glutamate content caused by an activation of glutaminase (EC 3.5.1.2).
| S-EPMC1153292 | biostudies-other
Project description:Rats were stressed by intravenous injection, tail-warming or moderate restraint for 30s, i.e. by stresses imposed by normal handling during experiment. Liver glutamate concentrations were greatly affected. The results were substantially the same in two varieties of rat (Wistar and Sprague-Dawley), in two laboratories, in experiments carried out by two sets of workers, and after all three stresses. The following detailed results refer to Wistar rats. 1. In starved rats at 20 degrees C and 30 degrees C and in post-absorptive rats at 20 degrees C stress by injection raised liver glutamate concentrations from 1.54, 1.57 and 1.88mumol/g wet wt. 30s after injection to 3.4, 2.7 and 3.6mumol/g wet wt. respectively a few minutes later. In starved rats at 20 degrees C the concentration then fell slowly to 2.3mumol/g wet wt., in starved rats at 30 degrees C it remained steady, and in post-absorptive rats at 20 degrees C it rose slowly to about 4.3mumol/g wet wt. The final values seemed fairly steady and corresponded to an ;alert' state. 2. In starved rats at 20 degrees C anaesthesia, with or without injection or cannulation during it, raised glutamate concentrations to the ;alert' values, which were maintained for 2-3h. 3. Liver alanine concentration in post-absorptive rats initially fell from 1.5 to 0.8mumol/g, and then stayed fairly constant. 4. Aspartate and glutamine concentrations altered only in starved rats, and proportionately much less than those of glutamate. 5. The necessity for knowing the time-dependence of glutamate concentrations after experimental handling is emphasized. 6. There is no wholly satisfactory explanation of the observations.
| S-EPMC1178181 | biostudies-other
Project description:High concentrations of urea were shown to induce a paradoxical regulatory volume decrease response with K(+) channel opening and subsequent hepatocyte shrinkage (Hallbrucker, C., vom Dahl, S., Ritter, M., Lang, F., and Häussinger, D. (1994) Pflügers Arch. 428, 552-560), although the hepatocyte plasma membrane is thought to be freely permeable to urea. The underlying mechanisms remained unclear. As shown in the present study, urea (100 mmol/liter) induced within 1 min an activation of ?(1) integrins followed by an activation of focal adhesion kinase, c-Src, p38(MAPK), extracellular signal-regulated kinases, and c-Jun N-terminal kinase. Because ?(5)?(1) integrin is known to act as a volume/osmosensor in hepatocytes, which becomes activated in response to hepatocyte swelling, the findings suggest that urea at high concentrations induces a nonosmotic activating perturbation of this osmosensor, thereby triggering a volume regulatory K(+) efflux. In line with this, similar to hypo-osmotic hepatocyte swelling, urea induced an inhibition of hepatic proteolysis, which was sensitive to p38(MAPK) inhibition. Molecular dynamics simulations of a three-dimensional model of the ectodomain of ?(5)?(1) integrin in water, urea, or thiourea solutions revealed significant conformational changes of ?(5)?(1) integrin in urea and thiourea solutions, in contrast to the simulation of ?(5)?(1) in water. These changes lead to an unbending of the integrin structure around the genu, which may suggest activation, whereas the structures of single domains remained essentially unchanged. It is concluded that urea at high concentrations affects hepatic metabolism through direct activation of the ?(5)?(1) integrin system.
| S-EPMC2937967 | biostudies-literature
Project description:Switching in acidosis of hepatic nitrogen disposal from urea synthesis to NH4+ and net glutamine production was demonstrated in the isolated perfused livers of starved male Wistar rats. Lactate was preferred to glucose as the substrate for the carbon skeleton of glutamine synthesized over the pH range 6.9-7.5. This is necessary if the switch away from a proton-producing process (ureagenesis) in acidosis is to constitute an acid-base regulating system intrinsic to the liver. Glutamine balance shifted with pH from marked net uptake to small net output under acidotic conditions (pH 7.5-6.9), an effect due solely to a decrease in glutamine uptake. NH4+ uptake by the liver had a linear relationship with pH, being markedly decreased in acidosis because glutamine synthesis was insufficient to compensate for the decreased incorporation into urea. Animals rendered chronically acidotic showed a lower central venous plasma urea concentration and a raised NH4+ concentration, but their livers synthesized no more glutamine when perfused at an acidotic pH than did normal livers. We conclude that perivenous hepatocytes may not be efficient scavengers of NH4+ ions, which must be partly disposed of elsewhere by non-proton-generating pathways if inhibition of ureagenesis is to represent a hepatic acid-base regulating system.
| S-EPMC1151404 | biostudies-other
Project description:1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.
| S-EPMC1178180 | biostudies-other
Project description:The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.
| S-EPMC1223758 | biostudies-other