Flux through glycine cleavage system in isolated hepatocytes: effects of glucagon, cAMP, and calcium

1990 ◽  
Vol 68 (2) ◽  
pp. 543-546 ◽  
Author(s):  
Markandeya Jois ◽  
Beatrice Hall ◽  
Vaughn M. Collett ◽  
John T. Brosnan
Keyword(s):  
Acid Metabolism ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Glycine Cleavage System ◽  
Isolated Rat Hepatocytes ◽  
Maximal Stimulation ◽  
Glycine Cleavage ◽  
Camp Levels ◽  
Stimulation Of

The hepatic glycine cleavage system (GCS) is the principal route for the metabolism of glycine in mammals. Flux through the GCS in isolated rat hepatocytes was stimulated by about 100% by glucagon (10−7 M), forskolin (10−4 M), and dibutyryl cAMP (10−4 M). The stimulation of flux through the GCS by these agents was accompanied by marked elevation of cellular cAMP levels. A significant correlation was observed between increased cellular cAMP levels induced by glucagon and stimulation of flux through the GCS by glucagon. Exclusion of calcium from the incubation medium reduced the basal flux by 38%, but did not affect the degree of stimulation of flux through the GCS by glucagon. A single intraperitoneal injection of glucagon to rats prior to isolation of hepatocytes resulted in a 76% stimulation of flux through the GCS. These hepatocytes with stimulated flux through the GCS showed reduced sensitivity for further stimulation by glucagon. Half-maximal stimulation of flux through the GCS occurred at 3.8 ± 1.1 and 8.5 ± 1.4 nM glucagon in hepatocytes isolated from control and glucagon-injected rats, respectively. We conclude that cAMP is involved in the regulation of flux through the GCS by gluagon.Key words: amino acid, metabolism, liver, mitochondria, hormones.

Threonine metabolism in isolated rat hepatocytes

2001 ◽  
Vol 281 (6) ◽  
pp. E1300-E1307 ◽  
Author(s):  
James D. House ◽  
Beatrice N. Hall ◽  
John T. Brosnan
Keyword(s):  
Potent Inhibitor ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Keto Acid ◽  
Glycine Cleavage System ◽  
Isolated Rat Hepatocytes ◽  
Threonine Dehydratase ◽  
Threonine Dehydrogenase ◽  
Glycine Cleavage

The removal of the 1-carbon of threonine can occur via threonine dehydrogenase or threonine aldolase, this carbon ending up in glycine to be liberated by the mitochondrial glycine cleavage system and producing CO2. Alternatively, in the threonine dehydratase pathway, the 1-carbon ends up in α-ketobutyrate, which is oxidized in the mitochondria to CO2. Rat hepatocytes, incubated in Krebs-Henseleit medium, were incubated with 0.5 mMl-[1-14C]threonine, and14CO2 production was measured. Added glycine (0.3 mM) marginally suppressed threonine oxidation. Cysteamine (0.5 mM), a potent inhibitor of the glycine cleavage system, reduced threonine oxidation to 65% of controls. However, α-cyanocinnamate (0.5 mM), a competitive inhibitor of mitochondrial α-keto acid uptake, reduced threonine oxidation to 35% of controls. These data provided strong evidence that ∼65% of threonine oxidation occurs through the glycine-independent threonine dehydratase pathway. Glucagon (10−7 M) increased threonine oxidation and stimulated threonine uptake by these cells. In summary, the majority of threonine oxidation occurs through the threonine dehydratase pathway in rat hepatocytes, and threonine oxidation is increased by glucagon, which also increases threonine's transport.

scholarly journals Insulin stimulates synthesis of soluble proteins in isolated rat hepatocytes

1980 ◽  
Vol 190 (3) ◽  
pp. 615-619 ◽  
Author(s):  
R L Clark ◽  
R J Hansen
Keyword(s):  
Protein Synthesis ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Cellular Protein ◽  
Soluble Proteins ◽  
Leucine Incorporation ◽  
Isolated Rat Hepatocytes ◽  
Hepatic Protein Synthesis ◽  
Isolated Rat ◽  
Stimulation Of

The incorporation of [3H]leucine into soluble cellular protein was measured in isolated hepatocytes at extracellular leucine concentrations ranging from 0.15 to 20.0 mM. Insulin caused a 12—15% stimulation of [3H]leucine incorporation in the presence of high extracellular leucine concentrations. It is concluded that insulin causes a small but significant increase in the rate of hepatic protein synthesis.

scholarly journals Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes

1999 ◽  
Vol 341 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Roland B. GREGORY ◽  
Robert A. WILCOX ◽  
Leise A. BERVEN ◽  
Nicole C. R. VAN STRATEN ◽  
Gijs A. VAN DER MAREL ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Cytoskeleton ◽  
Cytochalasin B ◽  
Rat Hepatocytes ◽  
Small Region ◽  
Cortical Actin ◽  
Isolated Rat Hepatocytes ◽  
Maximal Stimulation ◽  
Adenophostin A ◽  
Stimulation Of

The roles of a subregion of the endoplasmic reticulum (ER) and the cortical actin cytoskeleton in the mechanisms by which Ins(1,4,5)P3 induces the activation of store-operated Ca2+ channels (SOCs) in isolated rat hepatocytes were investigated. Adenophostin A, a potent agonist at Ins(1,4,5)P3 receptors, induced ER Ca2+ release and the activation of Ca2+ inflow. The concentration of adenophostin A that gave half-maximal stimulation of Ca2+ inflow (10 nM) was substantially lower than that (20 nM) which gave half-maximal ER Ca2+ release. A low concentration of adenophostin A (approx. 13 nM) caused near-maximal stimulation of Ca2+ inflow but only 20% of maximal ER Ca2+ release. Similar results were obtained using another Ins(1,4,5)P3-receptor agonist, 2-hydroxyethyl-α-D-glucopyranoside 2,3′,4′-trisphosphate. Anti-type-1 Ins(1,4,5)P3-receptor monoclonal antibody 18A10 inhibited vasopressin-stimulated Ca2+ inflow but had no observable effect on vasopressin-induced ER Ca2+ release. Treatment with cytochalasin B at a concentration that partially disrupted the cortical actin cytoskeleton inhibited Ca2+ inflow and ER Ca2+ release induced by vasopressin by 73 and 45%, respectively. However, it did not substantially affect Ca2+ inflow and ER Ca2+ release induced by thapsigargin or 13 nM adenophostin A, intracellular Ca2+ release induced by ionomycin or Ins(1,4,5)P3P4(5)-1-(2-nitrophenyl)ethyl ester [‘caged’ Ins(1,4,5)P3] or basal Ca2+ inflow. 1-(5-Chloronaphthalene-1-sulphonyl)homopiperazine, HCl (ML-9), an inhibitor of myosin light-chain kinase, also inhibited vasopressin-induced Ca2+ inflow and ER Ca2+ release by 53 and 44%, respectively, but had little effect on thapsigargin-induced Ca2+ inflow and ER Ca2+ release. Neither cytochalasin B nor ML-9 inhibited vasopressin-induced Ins(1,4,5)P3 formation. It is concluded that the activation of SOCs in rat hepatocytes induced by Ins(1,4,5)P3 requires the participation of a small region of the ER, which is distinguished from other regions of the ER by a different apparent affinity for Ins(1,4,5)P3 analogues and is associated with the plasma membrane through the actin skeleton. This conclusion is discussed briefly in relation to current hypotheses for the activation of SOCs.

scholarly journals Studies on the assembly of cytochrome oxidase in isolated rat hepatocytes

1982 ◽  
Vol 204 (1) ◽  
pp. 239-245 ◽  
Author(s):  
A Wielburski ◽  
S Kuźela ◽  
B D Nelson
Keyword(s):  
Cytochrome Oxidase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Small Scale ◽  
Pulse Label ◽  
Isolated Rat Hepatocytes ◽  
Sequential Event ◽  
Triton X ◽  
Isolated Liver

1. The assembly of rat liver cytochrome oxidase was studied in isolated hepatocytes and isolated liver mitochondria labelled with L-[35S]methionine. 2. Labelled subunits II and III appeared in the immunoabsorbed holoenzyme within minutes after the initiation of a pulse label. In contrast, labelled subunit I appeared in immunoabsorbed holoenzyme only after a subsequent 2 h chase or after an additional 2 h of labelling. Subunit I was heavily labelled, however, in intact mitochondria after 10 min. 3. A similar pattern of labelling was observed in holo-cytochrome oxidase which was chemically isolated by a small scale procedure adapted for this purpose. The appearance of subunit I in the holoenzyme was delayed for 1.5-2 h after a 60 min pulse with labelled methionine. 4. Incubation of hepatocytes for 4 h in the presence of cycloheximide had no effect on the labelling pattern described above. 5. Methods were developed in which newly translated, presumably unassembled, subunits of cytochrome oxidase could be separated from the holoenzyme by fractionation in Triton X-114. Short-term pulse experiments indicate that subunits II and III are associated with the holoenzyme fraction immediately after their completion, whereas subunit I is not. 6. The data are consistent with a model in which cytochrome oxidase assembly is viewed as an ordered and sequential event.

scholarly journals Effect of calcium ions on ethanol oxidation and drug glucuronidation in isolated hepatocytes

1979 ◽  
Vol 184 (3) ◽  
pp. 709-711 ◽  
Author(s):  
B Andersson ◽  
D P Jones ◽  
S Orrenius
Keyword(s):  
Drug Metabolism ◽  
Calcium Ions ◽  
Ethanol Oxidation ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Physiological Concentration ◽  
Isolated Rat Hepatocytes ◽  
Similar Dependence ◽  
Isolated Rat ◽  
Stimulation Of

The effect of extracellular Ca2+ concentration on ethanol oxidation and drug metabolism was studied in isolated rat hepatocytes. Both ethanol oxidation and drug glucuronidation showed similar dependence upon Ca2+, which was a stimulation of activity as Ca2+ was increased to physiological concentration, and inhibition at higher concentration.

Insulin binding and action in isolated rat hepatocytes: effect of obesity and fasting

1982 ◽  
Vol 243 (3) ◽  
pp. E240-E245 ◽  
Author(s):  
H. J. Frank ◽  
M. B. Davidson
Keyword(s):  
Insulin Response ◽  
Rat Hepatocytes ◽  
Insulin Binding ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Obese Rats ◽  
Glucose Incorporation ◽  
Receptor Number ◽  
Insulin Responses ◽  
Stimulation Of

Insulin binding and action on [14C]glucose incorporation into glycogen were studied in freshly isolated hepatocytes from control, obese, and 48-h fasted-obese rats. Tracer 125I-insulin binding was reduced in the obese animals from 20.8 +/- 1.3% in the controls of 15.9 +/- 0.8% (mean +/- SE). The change in binding was due to a decrease in receptor number with no change in affinity. Fasting the obese animal for 48 h restored the tracer 125I-insulin binding and the receptor number to control levels. Physiologic concentrations of insulin caused an 86% stimulation of net [14C]glucose incorporation into glycogen above the basal level of 9.0 +/- 1.0 nmol incorporated . 10(6) cells-1 . h-1 with an ED50 of 4.0 ng/ml. In obese (greater than 500 g) animals the maximum insulin response measured by percent increase above basal (49%) was reduced, and the ED50 (5.8 ng/ml) was increased. When the obese animals were fasted, the basal and maximum insulin responses were further depressed, but the ED50 was restored to control levels. In conclusion, hepatocytes from obese animals show both a receptor and postreceptor defect. Fasting the animals restores the receptor status to normal, but the postreceptor metabolic defect remains.

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes

1989 ◽  
Vol 37 (1) ◽  
pp. 23-31 ◽  
Author(s):  
M. Marinovich ◽  
L.M. Flaminio ◽  
M. Papagni ◽  
C.L. Galli
Keyword(s):  
Arachidonic Acid ◽  
Acid Metabolism ◽  
Rat Hepatocytes ◽  
Arachidonic Acid Metabolism ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat ◽  
Stimulation Of

scholarly journals α-adrenergic stimulation of respiration in isolated rat hepatocytes

1983 ◽  
Vol 210 (3) ◽  
pp. 867-873 ◽  
Author(s):  
A Binet ◽  
M Claret
Keyword(s):  
Respiration Rate ◽  
Rat Hepatocytes ◽  
Adrenergic Stimulation ◽  
Isolated Rat Hepatocytes ◽  
Cell Responses ◽  
Maximal Stimulation ◽  
Lag Period ◽  
Dose Dependent ◽  
Isolated Rat ◽  
Stimulation Of

1. The alpha-adrenergic agonists noradrenaline (in the presence of beta-blocker) and phenylephrine cause a transient stimulation of the respiration in isolated rat hepatocytes. After a lag period of 12s, this activation first attains its maximal value (+24%) for about 1 min and then falls to a sustained value (+15%). The effect is blocked by the alpha-antagonists phenoxybenzamine and phentolamine. It is dose-dependent, with an half-maximal stimulation by 16 nM-noradrenaline, which is similar to that found for other cell responses to the hormone. 2. Vasopressin and ATP, which in common with alpha-agonists are believed to increase intracellular [Ca2+], induce similar activation in the respiration rate. 3. The alpha-adrenergic-mediated respiration depends on extracellular Ca2+. The activation is decreased or abolished when extracellular [Ca2+] is decreased by adding EGTA, or when the Ca2+ antagonists Mn2+ and La3+ are present in the incubation medium. 4. It is suggested that the activation of the mitochondrial respiration rate results from the increase in cytosolic Ca2+ concentration, presumably via Ca2+ influx or Ca2+ release from the plasma membrane or endoplasmic reticulum.

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