Glutamine metabolism in isolated incubated adipocytes of the rat.
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ABSTRACT:
SUBMITTER: Kowalchuk JM
PROVIDER: S-EPMC1148763 | biostudies-other | 1988 Feb
REPOSITORIES: biostudies-other
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Project description:The mechanisms by means of which phenylephrine stimulates glutamine metabolism were studied in isolated rat hepatocytes. In the first 2 min after phenylephrine addition there was a rapid fall in the concentrations of intracellular 2-oxoglutarate and glutamate, presumably owing to activation of 2-oxoglutarate dehydrogenase. This was followed 2-3 min later by activation of glutaminase and by increases in glutamate and 2-oxoglutarate. Activation of glutaminase by phenylephrine was due to direct stimulation of the enzyme rather than to reversal of inhibition by the decrease in 2-oxoglutarate and glutamate. The stimulation of glutaminase by phenylephrine is partly due to an increase in the affinity of the enzyme for ammonia, its essential activator. It is concluded that stimulation of steady-state flux through the pathway from glutamine to glucose and urea can only be achieved by stimulation of glutaminase, the first enzyme in the pathway.
| S-EPMC1152637 | biostudies-other
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 facilitative glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in adipocytes and muscles, and the participation of GLUT4 in the pathogenesis of various clinical conditions associated with obesity, visceral fat accumulation and insulin resistance has been proposed. Glucose uptake by some members of the GLUT family, mainly GLUT1, is inhibited by flavonoids, the natural polyphenols present in fruits, vegetables and wine. Therefore it is of interest to establish if these polyphenolic compounds present in the diet, known to be effective antioxidants but also endowed with several other biological activities such as protein-tyrosine kinase inhibition, interfere with GLUT4 function. In the present study, we show that three flavonoids, quercetin, myricetin and catechin-gallate, inhibit the uptake of methylglucose by adipocytes over the concentration range of 10-100 microM. These three flavonoids show a competitive pattern of inhibition, with K(i)=16, 33.5 and 90 microM respectively. In contrast, neither catechin nor gallic acid inhibit methylglucose uptake. To obtain a better understanding of the interaction among GLUT4 and flavonoids, we have derived a GLUT4 three-dimensional molecular comparative model, using structural co-ordinates from a GLUT3 comparative model and a mechanosensitive ion channel [PDB (Protein Data Bank) code 1MSL] solved by X-ray diffraction. On the whole, the experimental evidence and computer simulation data favour a transport inhibition mechanism in which flavonoids and GLUT4 interact directly, rather than by a mechanism related to protein-tyrosine kinase and insulin signalling inhibition. Furthermore, the results suggest that GLUT transporters are involved in flavonoid incorporation into cells.
| S-EPMC1134865 | biostudies-literature
Project description:1. Adipocytes isolated from epididymal fat-pads of fed rats were incubated with different concentrations of glucagon, insulin, adrenaline and adenosine deaminase, and the effects of these agents on the ;initial' activity of acetyl-CoA carboxylase in the cells were studied. 2. Glucagon (at concentrations between 0.1 and 10nm) inhibited acetyl-CoA carboxylase activity. Maximal inhibition was approx. 70% of the ;control' activity in the absence of added hormone, and the concentration of hormone required for half-maximal inhibition was 0.3-0.5nm-glucagon. 3. Incubation of cells with adenosine deaminase resulted in a similar inhibition of acetyl-CoA carboxylase activity. Preincubation of adipocytes with adenosine deaminase did not alter either the sensitivity of carboxylase activity to increasing concentrations of glucagon or the maximal extent of inhibition. 4. Adrenaline inhibited acetyl-CoA carboxylase to the same extent as glucagon. Preincubation of the cells with glucagon did not alter the sensitivity of enzyme activity to adrenaline or the degree of maximal inhibition. 5. Insulin activated the enzyme by 70-80% of ;control' activity. Preincubation of the cells with glucagon did not alter the concentration of insulin required to produce half the maximal stimulatory effect (about 12muunits of insulin/ml). The effects of insulin and glucagon appeared to be mediated completely independently, and were approximately quantitatively similar but opposite. These characteristics resulted in the mutual cancellation of the effects of the two hormones when they were both present at equally effective concentrations. 6. The implications of these findings with regard to current concepts about the mechanism of regulation of acetyl-CoA carboxylase and to the regulation of the enzyme in vivo are discussed.
| S-EPMC1154031 | biostudies-other
Project description:The metabolism of glutamine in resting and concanavalin-A-stimulated lymphocytes was investigated. In incubated lymphocytes isolated from rat mesenteric lymph nodes, the rates of oxygen and glutamine utilization and that of aspartate production were approximately linear with respect to time for 60 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 90 min. The major end products of glutamine metabolism were glutamate, aspartate and ammonia: the carbon from glutamine may contribute about 30% to respiration. When both glucose and glutamine were presented to the cells, the rates of utilization of both substances increased. Evidence was obtained that the stimulation of glycolysis by glutamine could be due, in part, to an activation of 6-phosphofructokinase. Starvation of the donor animal increased the rate of glutamine utilization. The phosphoenolpyruvate carboxykinase inhibitor mercaptopicolinate decreased the rate of glutamine utilization by 28%; the rates of accumulation of glutamate and ammonia were decreased, whereas those of lactate, aspartate and malate were increased. The mitogen concanavalin A increased the rate of glutamine utilization (by about 51%). The rate of [3H]thymidine incorporation into DNA caused by concanavalin A in cultured lymphocytes was very low in the absence of glutamine; it was increased about 4-fold at 1 microM-glutamine and was maximal at 0.3 mM-glutamine; neither other amino acids nor ammonia could replace glutamine.
| S-EPMC1153161 | biostudies-other
Project description:The metabolism of glucose and glutamine in freshly prepared resting and concanavalin A-stimulated rat thymocytes was studied. Concanavalin A addition enhanced uptake of both glucose and glutamine and led to an increase in oxidative degradation of both substrates to CO2. With variously labelled [14C]glucose, it was shown that the pathways of glucose dissimilation were equally stimulated by the mitogen. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused an increase in the oxidation of pyruvate as judged by the enhanced release of 14CO2 from [2-14C]-, [3,4-14C]- and [6-14C]-glucose. Addition of glutamine did not affect glucose metabolism. The major end products of glutamine metabolism by resting and mitogen-stimulated rat thymocytes were glutamate, aspartate, CO2 and NH3. Virtually no lactate was formed from glutamine. Concanavalin A enhanced the formation of all end products except glutamate, indicating that more glutamine was metabolized beyond the stage of glutamate in the mitogen-activated cells. Addition of glucose caused a significant decrease in the rates of glutamine utilization and conversion into aspartate and CO2 in the absence and in the presence of concanavalin A. In the presence of glucose, almost all nitrogen of the metabolized glutamine was accounted for as NH3 released via the glutaminase and/or glutamate dehydrogenase reactions. In the absence of glucose, part (18%) of the glutamine nitrogen was metabolized by the resting and to a larger extent (38%) by the mitogen-stimulated thymocytes via a transaminase or amidotransferase reaction.
| S-EPMC1144061 | biostudies-other
Project description:Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitro PanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation.
| S-EPMC5014359 | biostudies-literature
Project description:Aristolochic acids are natural nitro-compounds found globally in the plant genus Aristolochia that have been implicated in the severe illness in humans termed aristolochic acid nephropathy (AAN). Aristolochic acids undergo nitroreduction, among other metabolic reactions, and active intermediates arise that are carcinogenic. Previous experiments with rats showed that aristolochic acid I (AA-I), after oral administration or injection, is subjected to detoxication reactions to give aristolochic acid Ia, aristolactam Ia, aristolactam I, and their glucuronide and sulfate conjugates that can be found in urine and feces. Results obtained with whole rats do not clearly define the role of liver and kidney in such metabolic transformation. In this study, in order to determine the specific role of the kidney on the renal disposition of AA-I and to study the biotransformations suffered by AA-I in this organ, isolated kidneys of rats were perfused with AA-I. AA-I and metabolite concentrations were determined in perfusates and urine using HPLC procedures. The isolated perfused rat kidney model showed that AA-I distributes rapidly and extensively in kidney tissues by uptake from the peritubular capillaries and the tubules. It was also established that the kidney is able to metabolize AA-I into aristolochic acid Ia, aristolochic acid Ia O-sulfate, aristolactam Ia, aristolactam I, and aristolactam Ia O-glucuronide. Rapid demethylation and sulfation of AA-I in the kidney generate aristolochic acid Ia and its sulfate conjugate that are voided to the urine. Reduction reactions to give the aristolactam metabolites occur to a slower rate. Renal clearances showed that filtered AA-I is reabsorbed at the tubules, whereas the metabolites are secreted. The unconjugated metabolites produced in the renal tissues are transported to both urine and perfusate, whereas the conjugated metabolites are almost exclusively secreted to the urine.
| S-EPMC3276251 | biostudies-literature
Project description:The effects of dithiothreitol on basal glucose oxidation, hormone-induced lipolysis and insulin receptors in isolated rat adipocytes were studied. Dithiothreitol produced a dose-dependent stimulation of basal glucose oxidation and inhibition of adrenaline-induced lipolysis. Dithiothreitol also inhibited corticotropin-induced lipolysis, but failed to inhibit dibutyryl cyclic AMP-induced lipolysis. Dithiothreitol did not inhibit the binding of the beta-adrenergic antagonist [3H]dihydroalprenolol to adipocytes. Neither catalase (100 micrograms/ml) nor EDTA (2 mM) abolished the antilipolytic effect of dithiothreitol. Treatment of isolated adipocytes with 1 mM-dithiothreitol for 20 min at 37 degrees C also caused stimulation of basal glucose oxidation and inhibition of adrenaline-induced lipolysis. A Scatchard plot of insulin binding to control adipocytes was curvilinear. However, treatment of cells with 1 mM-dithiothreitol decreased the curvilinearity of the plot, indicating that only a low-affinity state of the insulin receptors exists in the dithiothreitol-treated adipocytes. These findings suggest that the insulin-like activities of dithiothreitol are mediated through the interaction of dithiothreitol with insulin receptors.
| S-EPMC1163552 | biostudies-other
Project description:Altered metabolism is a hallmark of cancer cells. Tumor cells rewire their metabolism to support their uncontrolled proliferation by taking up nutrients from the microenvironment. The amino acid glutamine is a key nutrient that fuels biosynthetic processes including ATP generation, redox homeostasis, nucleotide, protein, and lipid synthesis. Glutamine as a precursor for the neurotransmitter glutamate, and plays a critical role in the normal functioning of the brain. Brain tumors that grow in this glutamine/glutamate rich microenvironment can make synaptic connections with glutamatergic neurons and reprogram glutamine metabolism to enable their growth. In this review, we examine the functions of glutamate/glutamine in the brain and how brain tumor cells reprogram glutamine metabolism. Altered glutamine metabolism can be leveraged to develop non-invasive imaging strategies and we review these imaging modalities. Finally, we examine if targeting glutamine metabolism could serve as a therapeutic strategy in brain tumors.
| S-EPMC6893651 | biostudies-literature