scholarly journals Stimulation of mitochondrial functions by glucagon treatment. Evidence that effects are not artifacts of mitochondrial isolation

1983 ◽  
Vol 210 (3) ◽  
pp. 819-827 ◽  
Author(s):  
C B Jensen ◽  
F D Sistare ◽  
H C Hamman ◽  
R C Haynes
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Treatment Result ◽  
Direct Result ◽  
Differential Centrifugation ◽  
Hormonal Stimulation ◽  
Pyruvate Carboxylation ◽  
Mitochondrial Functions ◽  
Liver Homogenates ◽  
Citrulline Synthesis ◽  
Stimulation Of

(1) Activation of rat liver mitochondrial functions following glucagon treatment was demonstrated in mitochondria that had not been isolated by the conventional technique of differential centrifugation and washing in sucrose solutions. Crude liver homogenates in 0.3 M-sucrose or 0.15 M-KCl prepared from rats treated with glucagon showed stimulation of State-3 and uncoupled respiration, carboxylation of pyruvate, and citrulline synthesis comparable with those previously reported in isolated mitochondria. (2) During the isolation procedure of mitochondria the hormonal stimulations of pyruvate carboxylation and citrulline formation were shown not to be enhanced by sequential washing. (3) Mitochondria isolated from glucagon-treated rats by differential centrifugation and washing in 0.3 M-mannitol/1 mM-EGTA, pH 7.0, exhibited a mean rate of citrulline synthesis that was greater than twice that of the control. Liver homogenates prepared in 0.3 M-sucrose or 0.3 M-mannitol showed identical rates of State-3 respiration and percentage stimulations of respiration by glucagon treatment. (4) Addition of glucagon led to a rapid accumulation of malate and aspartate and decreased the amounts of glutamate and citrate in isolated hepatocytes incubated with L-lactate. When gluconeogenesis was inhibited at the phosphoenolpyruvate carboxykinase (EC 4.1.1.32) reaction these phenomena were accentuated, lending support to the interpretation that they are the direct result of stimulation of carboxylation and oxidation reactions in the mitochondria. These results do not support the proposal [Siess, Fahimi & Wieland (1981) Hoppe-Seyler's Z. Physiol. Chem. 362. 1643-1651] that the mitochondrial effects of glucagon treatment result from a stabilization of mitochondria to detrimental effects of sucrose during their isolation. (5) The mean hormonal stimulation of pyruvate carboxylation in mitochondria isolated in 0.3 M-sucrose was shown to be approx. 2.5-fold when assayed either at 37 degrees C or 25 degrees C. In contrast, on the basis of similar experiments, Siess et al. (1981) concluded that the effects of glucagon on hepatic mitochondria are not characteristic of a true hormonal stimulation. Our data indicate this conclusion to be unjustified.

scholarly journals Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes

1983 ◽  
Vol 212 (2) ◽  
pp. 417-426 ◽  
Author(s):  
E H Allan ◽  
A B Chisholm ◽  
M A Titheradge
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Co2 Fixation ◽  
Hormone Action ◽  
Acute Effects ◽  
Polyene Antibiotic ◽  
Hormonal Stimulation ◽  
Pyruvate Carboxylation ◽  
Isolated Mitochondria ◽  
Stimulation Of

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.

scholarly journals The stimulation of mitochondrial pyruvate carboxylation after dexamethasone treatment of rats

1984 ◽  
Vol 219 (1) ◽  
pp. 107-115 ◽  
Author(s):  
A D Martin ◽  
E H Allan ◽  
M A Titheradge
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Form ◽  
Specific Inhibitor ◽  
Hormone Treatment ◽  
Dexamethasone Treatment ◽  
Pyruvate Metabolism ◽  
Glucocorticoid Treatment ◽  
Pyruvate Carboxylation ◽  
Liver Homogenates ◽  
Stimulation Of

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.

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

1984 ◽  
Vol 222 (2) ◽  
pp. 379-387 ◽  
Author(s):  
A D Martin ◽  
M A Titheradge
Keyword(s):  
Adenine Nucleotides ◽  
Isolation Procedure ◽  
Mitochondrial Matrix ◽  
Pyruvate Metabolism ◽  
Pyruvate Carboxylation ◽  
Matrix Volume ◽  
The Matrix ◽  
Citrulline Synthesis ◽  
Stimulation Of

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.

scholarly journals Sub-micromolar concentrations of extramitochondrial Ca2+ stimulate the rate of citrulline synthesis by rat liver mitochondria

1989 ◽  
Vol 257 (1) ◽  
pp. 285-288 ◽  
Author(s):  
J D Johnston ◽  
M D Brand
Keyword(s):  
Rat Liver ◽  
Urea Cycle ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Hormonal Stimulation ◽  
Isolated Rat Liver ◽  
Citrulline Synthesis ◽  
Isolated Rat ◽  
Stimulation Of

1. In the presence of physiological concentrations of Na+ and Mg2+, the rate of citrulline synthesis by isolated rat liver mitochondria respiring on a range of substrates was stimulated by up to 60% when the extramitochondrial Ca2+ concentration was raised from 130 pM to 770 nM. 2. Our findings suggest that hormonal stimulation of the urea cycle may be mediated by Ca2+.

Secretagogue-induced changes in subcellular Ca2+ distribution in isolated pancreatic acini

1981 ◽  
Vol 240 (2) ◽  
pp. G130-G140
Author(s):  
R. L. Dormer ◽  
J. A. Williams
Keyword(s):  
Atomic Absorption Spectrometry ◽  
High Speed ◽  
Microsomal Fraction ◽  
Absorption Spectrometry ◽  
Subcellular Fractionation ◽  
Differential Centrifugation ◽  
Zymogen Granules ◽  
Pancreatic Acini ◽  
Induced Changes ◽  
Stimulation Of

In a prior study, we demonstrated that pancreatic secretagogues increased both the uptake into and washout of 45Ca2+ from isolated mouse pancreatic acini. The net result of these processes was an initial fall in total acinar cell Ca2+ content. In the present study, we have employed subcellular fractionation of acini under conditions that minimized posthomogenization redistribution of Ca2+ in order to localize those organelles involved in intracellular Ca2+ fluxes. Homogenization and differential centrifugation of acini, preloaded with 45Ca2+ and subjected to a period of washout, showed that carbachol induced an increased loss of 45Ca2+ from all fractions isolated. The high-speed microsomal fraction lost 45Ca2+ to a greater extent than did whole acini; measurement of total Ca2+ by atomic absorption spectrometry showed a net loss of Ca2+ from this fraction. Purification of the lower-speed fractions indicated that carbachol increased 45Ca2+ exchange with both zymogen granules and mitochondria, but net Ca2+ levels in these organelles were unchanged. It was concluded that stimulation of pancreatic acini by carbachol results in the release of calcium from a microsomal compartment leading to a rise in cytoplasmic Ca2+, increased exchange with granule and mitochondrial Ca2+, and increased efflux of Ca2+ from the cell.

AN INCREASED RATE OF CHOLESTEROL BIOSYNTHESIS WITH MICROSOME FRACTIONS OF LIVER FROM KETOTIC GUINEA PIGS

1962 ◽  
Vol 40 (1) ◽  
pp. 1749-1762 ◽  
Author(s):  
F. Sauer
Keyword(s):  
Young Adult ◽  
Guinea Pigs ◽  
Cholesterol Synthesis ◽  
Cholesterol Biosynthesis ◽  
Maximum Activity ◽  
Sterol Synthesis ◽  
Differential Centrifugation ◽  
Microsome Fraction ◽  
Liver Homogenates ◽  
Increased Activity

Cholesterol synthesis was studied in liver fractions obtained by differential centrifugation from young, adult, and ketotic guinea pigs. Both 10,000 × g and 105,000 × g sediment was required for maximum activity. Incubations were carried out in the presence of appropriate liver fractions from young guinea pigs in order to overcome the low rates of cholesterol synthesis in liver homogenates from adult guinea pigs. Microsome fractions from ketotic hyperlipemic guinea pigs actively promoted sterol synthesis when incubated with mitochondria plus supernatant from young guinea pigs, while microsome fractions from adult controls (fed or starved) decreased the rate of sterol synthesis in the same incubation system. The results of this investigation indicate that microsomes from hyperlipemic ketotic guinea pigs do not have a block in cholesterol synthesis characteristic of microsomes from starved animals, and that this microsome fraction has increased activity of HMG-CoA2reductase, one of the key enzymes of cholesterol synthesis.

Cellular and molecular basis of increased ammoniagenesis in potassium deprivation

2011 ◽  
Vol 301 (5) ◽  
pp. F969-F978 ◽  
Author(s):  
Shaikh Abu Hossain ◽  
Farrukh A. Chaudhry ◽  
Kamyar Zahedi ◽  
Faraaz Siddiqui ◽  
Hassane Amlal
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Molecular Mechanisms ◽  
Intracellular Acidosis ◽  
Deficient Diet ◽  
Proximal Tubule Cells ◽  
Glutamine Transporter ◽  
Additive Increase ◽  
Acid Base Status ◽  
Low K ◽  
Stimulation Of

Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K+-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH4+ excretion increased significantly and correlated with a parallel decrease in urine K+ excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH4Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH4+ excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH4Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK1 in low-K+ medium did not affect NH4+ production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K+ deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH4+ production and decreased K+ excretion indicate that NH4+ synthesis and transport likely play an important role in renal K+ conservation during hypokalemia.

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