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

1983 ◽  
Vol 214 (2) ◽  
pp. 451-458 ◽  
Author(s):  
A B Chisholm ◽  
E H Allan ◽  
M A Titheradge
Keyword(s):  
Insulin Treatment ◽  
Second Messengers ◽  
Isolated Hepatocytes ◽  
Carboxylase Activity ◽  
Effector Molecules ◽  
Pyruvate Carboxylation ◽  
Isolated Mitochondria ◽  
Glucose Synthesis ◽  
Important Site ◽  
Stimulation Of

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.

scholarly journals FINE STRUCTURAL CHANGES IN RESPONSE TO HORMONAL STIMULATION OF THE PERFUSED CANINE PANCREAS

1965 ◽  
Vol 24 (3) ◽  
pp. 369-385 ◽  
Author(s):  
Atsushi Ichikawa
Keyword(s):  
Structural Changes ◽  
Detectable Effect ◽  
Secretory Product ◽  
Zymogen Granules ◽  
Hormonal Stimulation ◽  
Dog Blood ◽  
Secretory Products ◽  
Dog Pancreas ◽  
Stimulation Of

The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.

scholarly journals Intramitochondrial regulation of fatty acid β-oxidation occurs between flavoprotein and ubiquinone A role for changes in the matrix volume

1986 ◽  
Vol 239 (3) ◽  
pp. 559-565 ◽  
Author(s):  
A P Halestrap ◽  
J L Dunlop
Keyword(s):  
Fatty Acid ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Hormonal Stimulation ◽  
Terminal Electron Acceptor ◽  
Matrix Volume ◽  
The Matrix ◽  
Range Of Values ◽  
Stimulation Of

Rat liver mitochondria were incubated in media of different osmolarities and in the presence of various substrates. Rates of oxygen consumption and mitochondrial matrix volumes were measured in the presence and absence of ADP and uncoupler. Duroquinol oxidation was insensitive to matrix volume, whereas other substrates tested showed increased rates of oxidation when the matrix volume increased from 1.0 to 1.5 microliter/mg of protein; this is the range of values measured in situ [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. Palmitoylcarnitine, octanoate and butyrate oxidations were particularly sensitive to the matrix volume, increasing from negligible rates to maximal rates within this range. Swelling induced by K+ uptake also stimulated palmitoylcarnitine oxidation. A similar effect of volume on substrate oxidation was seen when ferricyanide in the presence or absence of ubiquinone-1 replaced oxygen as terminal electron acceptor. Measurement of flavoprotein reduction (A 460-480) demonstrated that the locus of the effect of matrix volume is between the electron-transfer flavoprotein and ubiquinone. It is suggested that volume-mediated regulation of fatty acid and proline oxidation may be an important component of the hormonal stimulation of their oxidation.

Regulation of total mitochondrial Ca2+ in perfused liver is independent of the permeability transition pore

1999 ◽  
Vol 276 (6) ◽  
pp. C1297-C1302 ◽  
Author(s):  
Ove Eriksson ◽  
Piero Pollesello ◽  
Erika Geimonen
Keyword(s):  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mitochondrial Content ◽  
Hormonal Stimulation ◽  
Intact Cells ◽  
Mitochondrial Energy Metabolism ◽  
Isolated Mitochondria ◽  
Rat Livers ◽  
Stimulation Of

Triggering of the permeability transition pore (PTP) in isolated mitochondria causes release of matrix Ca2+, ions, and metabolites, and it has been proposed that the PTP mediates mitochondrial Ca2+ release in intact cells. To study the role of the PTP in mitochondrial energy metabolism, the mitochondrial content of Ca2+, Mg2+, ATP, and ADP was determined in hormonally stimulated rat livers perfused with cyclosporin A (CsA). Stimulation of livers perfused in the absence of CsA with glucagon and phenylephrine induced an extensive uptake of Ca2+, Mg2+, and ATP plus ADP by the mitochondria, followed by a release on omission of hormones. In the presence of CsA, the PTP was fully inhibited, but neither the hormone-induced uptake of Ca2+, ATP, or ADP by mitochondria nor their release after washout of hormones was significantly changed. We conclude that the regulation of sustained changes in mitochondrial Ca2+content induced by hormonal stimulation is independent of the PTP.

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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