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M A Titheradge

Publications and source records attributed to M A Titheradge.

At least 37 records · Page 2Linked to original sources
PubMed1992

Effect of treatment in vivo of rats with bacterial endotoxin on fructose 2,6-bisphosphate metabolism and L-pyruvate kinase activity and flux in isolated liver cells.

The effect of treatment of rats with bacterial endotoxin on fructose 2,6-bisphosphate (Fru-2,6-P2) metabolism was investigated in isolated liver cells prepared from 18 h-starved animals. The results obtained support the hypothesis that a stimulation of 6-phosphofructo-1-kinase (PFK-1) activity and an inhibition of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) may be one mechanism underlying the inhibition of gluconeogenesis from lactate and pyruvate by endotoxin. We suggest that the stimulation of PFK-1 and inhibition of Fru-1,6-P2ase activity is the result of a 2-3-fold increase in Fru-2,6-P2. The latter is not due to changes in the total activity or phosphorylation state of the bifunctional 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase, but appears to be the result of a decrease in the cytosolic concentration of phosphoenolpyruvate (PEP), an inhibitor of PFK-2 activity. The effect of endotoxin is resistant to the presence of glucagon, which has comparable effects in cells prepared from both control and endotoxin-treated animals. The mechanism by which endotoxin treatment of the rat decreases PEP and gluconeogenesis remains to be established. However, it does not involve alterations in either the total activity or the phosphorylation state of pyruvate kinase, nor does it involve increased flux through this enzyme in the intact cell, which is in fact decreased in this model of septic shock. It is suggested that the decreased flux may result from a lower rate of formation of PEP, suggesting that the prime lesion in sepsis is an inhibition of one or more of the steps leading to PEP formation.

Animals↗
PubMed1990

The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain.

Our observations that opioid peptides have direct effects on islet insulin secretion and liver glucose production prompted a search for endogenous opiates and their receptors in these peripheral tissues. Mu-, delta- and kappa-receptor-active opiates were demonstrated in brain, pancreas and liver extracts by displacement studies using selective ligands for the three opiate receptor subtypes [( 3H][D-Ala2,MePhe4,Gly5-ol]enkephalin, [3H][D-Ala2,D-Leu5]enkephalin and [3H]dynorphin respectively). Receptor-active opiates in brain extracts exhibited a stronger preference for delta-opiate-receptor sites than for mu and kappa sites. Pancreatic extract opiates demonstrated a similar activity at mu and delta sites, but substantially less at kappa sites. Liver extracts displayed similar selectivity for all three sites. The affinities of the receptor-active opiates for mu-, delta- and kappa-receptor subtypes displayed a rank order of potency: brain much greater than pancreas greater than liver. Total immunoreactive beta-endorphin and [Met5]enkephalin levels in liver and hepatocytes were greater than those in brain. Immunoreactive [Met5]enkephalin levels in pancreas were similar to, but beta-endorphin levels were substantially higher than, those in brain. Delta and kappa opiate-binding sites of high affinity were identified in crude membrane preparations of islets of Langerhans, but no specific opiate-binding sites could be demonstrated in liver membrane preparations. Immunoreactive dynorphin and beta-endorphin were demonstrated by immunogold labelling in rat pancreatic islet cells. No positive staining of liver sections for opioids was observed. These results suggest that the tissue content of opiate-receptor-active compounds in the pancreas and the liver is very significant and could contribute to the regulation of normal blood glucose levels.

Animals↗
PubMed1989

[Leu]enkephalin stimulates carbohydrate metabolism in isolated hepatocytes and kidney tubule fragments by interaction with angiotensin II receptors.

The possibility that the effects of [Leu]enkephalin in vitro on hepatic carbohydrate metabolism are mediated by interaction with angiotensin II receptors has been examined. Preincubation of hepatocytes with either the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II or 10 mM-dithiothreitol abolished the ability of both angiotensin II and [Leu]enkephalin to increase phosphorylase a in hepatocytes prepared from fed rats. Dithiothreitol had no effect on the stimulation of phosphorylase in the presence of glucagon or phenylephrine, although it also inhibited the response to vasopressin. [Leu]enkephalin displaced specifically bound 125I-labelled angiotensin II from hepatic plasma membranes over a concentration range of 10(-7)-10(-5) M. This correlated with the dose-response required to stimulate phosphorylase activity in intact hepatocytes and suggests that the effects of the opioid peptides on carbohydrate metabolism in liver are the result of cross-reactivity of the peptides with angiotensin II receptors. Addition of 10(-5) M-[Leu]enkephalin to isolated kidney tubule fragments stimulated gluconeogenesis from 5 mM-pyruvate, the magnitude of stimulation being comparable to that by either angiotensin II or adrenaline. This effect of the opioid peptide was also abolished by pretreatment of the tubules with [Sar1,Ile8]angiotensin II, suggesting that the ability of [Leu]enkephalin to interact with angiotensin II receptors is not restricted to the liver, but may occur in other tissues where both receptors occur together.

Angiotensin II↗
PubMed1988

Comparison of the effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism.

The effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism are compared. Both peptides stimulated glycogenolysis as a result of an increase in phosphorylase a activity and enhanced glucose synthesis from [2-14C]pyruvate, although neither had any significant effect on pyruvate kinase activity. Although the magnitudes of the effects of both peptides on glycogenolysis were comparable and unaffected by the presence of insulin. [Leu]enkephalin proved to be more efficacious in enhancing gluconeogenesis, the response being comparable with that to glucagon. Both effectors decreased the intracellular concentration of cyclic AMP in hepatocytes when incubated under control conditions and after addition of sub-optimal concentrations of glucagon. This was correlated with the ability of the two peptides to inhibit both basal and hormone-stimulated adenylate cyclase activity in purified liver plasma membranes.

Adenylyl Cyclases↗
PubMed1986

The stimulation of glycogenolysis in isolated hepatocytes by opioid peptides.

The opioid peptides [Leu]enkephalin and dynorphin-(1-13) were shown to enhance glycogen breakdown when added directly to hepatocytes. This was the result of a concerted effect on the enzymes of glycogen metabolism, with a stimulation of glycogen phosphorylase activity and a simultaneous decrease in glycogen synthase I activity. The latter only became significant when the enzyme was activated by incubating the cells in presence of 20 mM- or 40 mM-glucose. The effect of the opioid peptides was independent of an increase in cyclic AMP or any change in the activity ratio of the cyclic AMP-dependent protein kinase and was abolished by depleting the cells of Ca2+. Both [Leu]enkephalin and dynorphin-(1-13) produced a significant decrease in cyclic AMP formation, suggesting that in liver, as in neuronal tissue, they may act by inhibiting adenylate cyclase activity.

Animals↗
PubMed1986

Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin.

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.

Animals↗
PubMed1985

The stimulation of glycogenolysis in isolated hepatocytes by opioid peptides.

Addition of the opioid peptides, [Leu]enkephalin and [Met]enkephalin, to isolated hepatocytes was shown to produce a stimulation of glycogenolysis comparable with that observed in the presence of maximal concentrations of glucagon, adrenaline or angiotensin. This stimulation was demonstrated to be the result of an activation of phosphorylase by a rapid Ca2+-dependent mechanism and was not decreased by the presence or either alpha- or beta-adrenergic antagonists, although it was dependent on the presence of the N-terminal tyrosine residue in the enkephalin molecule. It is suggested that this may be further evidence for specific opioid receptors in the liver. Addition of [Leu]enkephalin also inhibited lactate formation, indicating that the opioid peptides exert a concerted effect on hepatic carbohydrate metabolism to enhance glucose output. The transient nature of the effect of the enkephalins was shown to be the result of a rapid breakdown of the peptides in the incubation as a result of aminopeptidase activity, the initial product being the inactive des-tyrosine derivative.

Angiotensin II↗
PubMed1984

Stimulation of mitochondrial pyruvate metabolism and citrulline synthesis by dexamethasone. Effect of isolation and incubation media.

Hepatic mitochondria isolated in 0.3 M-sucrose or 0.3 M-mannitol from rats treated for 3h with dexamethasone displayed stimulated rates of pyruvate carboxylation and decarboxylation and citrulline synthesis when compared with organelles from control animals. Mitochondria isolated in mannitol also displayed elevated rates of pyruvate carboxylation and decarboxylation when compared with those isolated in sucrose, and this stimulation was shown to be independent of the lengthy isolation procedure. Citrulline synthesis proceeded at similar rates in mitochondria isolated in either sugar. The concentration of exchangeable adenine nucleotides was identical in mitochondria isolated in sucrose or mannitol, suggesting that those prepared in the former sugar are not more permeable to metabolites than those prepared in the latter. The matrix volume of mitochondria isolated in mannitol was greater than that of mitochondria isolated in sucrose, and the effect of mannitol on pyruvate metabolism was mimicked by swelling the organelles in hypo-osmotic sucrose. Measurements of the extra-matrix volume by using [14C]sucrose or [14C]mannitol suggest that mannitol can permeate mitochondria to a greater extent than can sucrose. The possibility that mannitol elicits its effect by entering the mitochondrial matrix and so initiating swelling is discussed.

Adenine Nucleotides↗
PubMed1984

The stimulation of mitochondrial pyruvate carboxylation after dexamethasone treatment of rats.

Treatment of rats for 3 h with dexamethasone was shown to stimulate both pyruvate carboxylation and decarboxylation in the subsequently isolated mitochondria. The effect of hormone treatment on pyruvate carboxylation was also apparent in liver homogenates assayed within minutes of killing the animal and was independent of the temperature at which the assay was performed, suggesting that it was not an artifact of the mitochondrial preparation procedure. The stimulation of both aspects of pyruvate metabolism in the intact organelle was independent of the induction of either pyruvate carboxylase or pyruvate dehydrogenase. Similarly, there was no change in the percentage of pyruvate dehydrogenase in the active form, indicating that the effect of steroid treatment on pyruvate oxidation was not via changes in the degree of phosphorylation of the enzyme. Adrenalectomizing the animals for a period of 14 days before the experiment had no effect on either parameter. Glucocorticoid treatment of the animals increased the rate of pyruvate uptake into the mitochondria, as measured by the titration of pyruvate metabolism with alpha-cyano-4-hydroxycinnamate, a specific inhibitor of the pyruvate translocator. It also increased the intramitochondrial concentrations of acetyl-CoA and ATP and led to an elevated [ATP]/[ADP] ratio within the mitochondria. It is suggested that both enzymes of pyruvate metabolism exist in the mitochondria under considerable restraint and that glucocorticoids act to relieve this restraint by alterations in substrate supply and the intramitochondrial concentrations of effector molecules.

Adrenalectomy↗
PubMed1984

Effect of treatment of rats with dexamethasone in vivo on gluconeogenesis and metabolite compartmentation in subsequently isolated hepatocytes.

Hepatocytes prepared from rats treated with dexamethasone for 2 or 3h and maintained in the presence of 10 microM-dexamethasone in the preparation and incubation buffers showed significantly elevated rates of gluconeogenesis compared with those prepared from control animals. Dexamethasone treatment also increased the sensitivity of the cells to glucagon and the catecholamines. Analysis of the concentrations of metabolites in the gluconeogenic pathway indicated that dexamethasone decreased the intracellular concentration of pyruvate and increased those of phosphoenolpyruvate, acetyl-CoA and citrate, suggesting a stimulation of the reaction(s) converting pyruvate into phosphoenolpyruvate. This was substantiated by analysis of the pattern of metabolites found in the mitochondrial compartment after digitonin fractionation of the cells. Inclusion of 3-mercaptopicolinate in the incubation enhanced the effect of the hormone on the distribution of metabolites. Thus, in the absence of an effect of the steroid at the level of phosphoenolpyruvate carboxykinase or pyruvate kinase, dexamethasone treatment still increased the formation of malate, aspartate and citrate from pyruvate, indicating a stimulation in the intact cell of pyruvate carboxylase. It is suggested that the stimulation of pyruvate carboxylase is a result of a general activation of mitochondrial function, with an increase in the intramitochondrial concentrations of acetyl-CoA and ATP, a decrease in glutamate and an enhanced intramitochondrial [ATP]/[ADP] ratio.

Animals↗
PubMed1983

The stimulation of glycogenolysis and gluconeogenesis in isolated hepatocytes by opioid peptides.

The opioid agonists [leucine]enkephalin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13)-peptide, but not morphine, stimulated the conversion of [2-14C]pyruvate into glucose and glycogenolysis when added directly to isolated hepatocytes. Naloxone produced a small but significant inhibition of both the basal and stimulated rate of incorporation of label into glucose but had no effect on the total glucose output by the cells. The effects of the opioid peptides were mediated by a cyclic AMP-independent mechanism.

Animals↗
PubMed1983

The stimulation of hepatic oxidative phosphorylation following dexamethasone treatment of rats.

The effect of short-term treatment of rats with the synthetic glucocorticoid, dexamethasone, on mitochondrial oxidative phosphorylation has been examined. Treatment of rats for 3 h increased the oxidative capacity of the subsequently isolated mitochondria such that they displayed increased uncoupled and State 3 rates of respiration with NAD-linked substrates, succinate or durohydroquinone. The oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine was unaffected. No change was apparent in the activity of a variety of dehydrogenase enzymes nor was there any increase in the mitochondrial content of cytochromes a, b, c1 or c. The uncoupler-dependent ATPase activity of the mitochondria was slightly enhanced following hormone treatment, but not the basal or the total ATPase activity measured in the presence of Triton X-100 plus Mg2+. The mitochondria prepared from dexamethasone-treated rats also displayed increased intramitochondrial concentrations of Mg2+, K+ and exchangeable adenine nucleotides but not Ca2+. It is suggested that the effect of glucocorticoids on mitochondrial respiration may be both the result of a direct activation of the respiratory chain within Complex III and an elevated intramitochondrial adenine nucleotide concentration. The evidence for the de novo synthesis of mitochondrial proteins which mediate the response remains inconclusive.

Animals↗
PubMed1983

Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment.

The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.

Acetyl Coenzyme A↗
PubMed1983

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes.

A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure.

Aminooxyacetic Acid↗
PubMed1980

The control of uncoupler-activated ATPase activity in rat liver mitochondria by adenine nucleotide transport. The effect of glucagon treatment.

Acute treatment of rats with glucagon increased the Vmax but did not change the Km (ATP) of uncoupler-activated ATPase in subsequently isolated hepatic mitochondria. The hormonal stimulation was evident in mitoplasts but not in submitochondrial particles nor after lysis of the mitochondria. The rate of Pi-ATP exchange of intact mitochondria was also increased by glucagon treatment. The hormonal stimulation of ATPase was dependent on concentration of the uncouplers, being absent at minimally effective concentrations while high concentrations inhibited the ATPase. Inhibitors of adenine nucleotide transport decreased ATPase activity without evidence of sigmoidicity in the response curves and produced linear Dixon plots indicating that the ATPase was limited by the rate of adenine nucleotide transport. Glucagon treatment did not change the number of binding sites for transport inhibitors. Glucagon stimulated the rate of transport of ATP as measured by accumulation of labeled nucleotide. This was found to be the consequence of an enlarged pool of exchangeable adenine nucleotides within mitochondria from glucagon-treated animals. This increase in mitochondrial nucleotides appears to explain a number of the effects of hormones on mitochondrial functions including the stimulation of uncoupler-activated ATPase activity.

Adenosine Triphosphatases↗