scholarly journals The glucagon-induced activation of pyruvate dehydrogenase in hepatocytes is diminished by 4β-phorbol 12-myristate 13-acetate. A role for cytoplasmic Ca2+ in dehydrogenase regulation

1987 ◽  
Vol 241 (3) ◽  
pp. 729-735 ◽  
Author(s):  
J M Staddon ◽  
R G Hansford
Keyword(s):  
Dehydrogenase Activity ◽  
Phorbol Ester ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Rat Hepatocytes ◽  
Pyruvate Kinase Activity ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Subsequent Addition ◽  
Isolated Rat

Phenylephrine, vasopressin and glucagon each increased the amount of active (dephospho) pyruvate dehydrogenase (PDHa) in isolated rat hepatocytes. Treatment with 4 beta-phorbol 12-myristate 13-acetate (PMA) opposed the increase in PDHa caused by both phenylephrine and glucagon, but had no effect on the response to vasopressin: PMA alone had no effect on PDHa. As PMA is known to prevent the phenylephrine-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and to diminish the increase [Ca2+]c caused by glucagon, while having no effect on the ability of vasopressin to increase [Ca2+]c, these data are consistent with the notion that in intact cells an increase in [Ca2+]c results in an increase in the mitochondrial free Ca2+ concentration, which in turn leads to the activation of PDH. In the presence of 2.5 mM-Ca2+, glucagon caused an increase in NAD(P)H fluorescence in hepatocytes. This increase is taken to reflect an enhanced activity of mitochondrial dehydrogenases. PMA alone had no effect on NAD(P)H fluorescence; it did, however, compromise the increase produced by glucagon. When the extracellular free [Ca2+] was decreased to 0.2 microM, glucagon could still increase NAD(P)H fluorescence. Vasopressin also increased fluorescence under these conditions; however, if vasopressin was added after glucagon, no further increase in fluorescence was observed. Treatment of the cells with PMA resulted in a smaller increase in NAD(P)H fluorescence on addition of glucagon: the subsequent addition of vasopressin now caused a further increase in fluorescence. Changes in [Ca2+]c corresponding to the changes in NAD(P)H fluorescence were observed, again supporting the idea that [Ca2+]c indirectly regulates intramitochondrial dehydrogenase activity in intact cells. PMA alone had no effect on pyruvate kinase activity, and the phorbol ester did not prevent the inactivation caused by glucagon. The latter emphasizes the different mechanisms by which the hormone influences mitochondrial and cytoplasmic metabolism.

scholarly journals Glucosamine-sensitive and -insensitive detritiation of [2-3H]glucose in isolated rat hepatocytes: a study of the contributions of glucokinase and glucose-6-phosphatase

1995 ◽  
Vol 308 (1) ◽  
pp. 23-29 ◽  
Author(s):  
E Van Schaftigen
Keyword(s):  
Exchange Reaction ◽  
Regulatory Protein ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Amino Sugar ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
In Situ Experiments ◽  
Glucose Phosphorylation ◽  
Isolated Rat

Glucosamine, a potent inhibitor of glucokinase (hexokinase IV or D), was used to estimate the contribution of this enzyme to glucose phosphorylation in freshly isolated rat hepatocytes and its sensitivity to fructose 6-phosphate in situ. Experiments with radiolabelled glucosamine indicated that this amino sugar, at concentrations of 5 or 40 mM, readily penetrated hepatocytes to reach in 1 min a total (i.e., glucosamine+metabolites) intracellular concentration equal to 0.8-1.2-fold its extracellular concentration. In marked contrast, N-acetylglucosamine barely penetrated the cells. The detritiation of [2-3H]glucose, used to estimate glucose phosphorylation in intact cells, was inhibited by glucosamine much more potently than by N-acetylglucosamine, half-maximal effects being reached at about 2.5 and 30 mM respectively. Extrapolation of the data indicated that about 12% of the detritiation was resistant to glucosamine. Dihydroxyacetone (10 mM), lactate (10 mM) + pyruvate (1 mM), and glucagon (1 microM) increased up to 8-fold the concentration of hexose 6-phosphates (glucose 6-phosphate+fructose 6-phosphate) and, against expectations, modestly decreased the detritiation rate measured in the absence of glucosamine. In the presence of 40 mM glucosamine, these agents increased the detritiation rate, which then positively correlated with the concentration of hexose 6-phosphates. This hexose 6-phosphates-dependent detritiation was sensitive to inhibition by vanadate, and was also catalysed by gel-filtered cell-free extracts, as well as by liver microsomes in the presence of phosphoglucoisomerase; it can be explained by an exchange reaction catalysed by glucose-6-phosphatase. When this exchange reaction is taken into account, it appears that the rate of glucose detritiation attributable to glucokinase decreases when the concentration of hexose 6-phosphates increases. This is in agreement with the known effect of fructose 6-phosphate to potentiate the inhibition of glucokinase by its regulatory protein.

scholarly journals Novel evidence for an ecto-phospholipid methyltransferase in isolated rat hepatocytes

1998 ◽  
Vol 330 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Françoise BONTEMPS ◽  
Georges VAN DEN BERGHE
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Methyl Group ◽  
Methyl Donor ◽  
External Face ◽  
Subsequent Addition ◽  
Isolated Rat

Phospholipids of isolated rat hepatocytes were labelled by preincubation with either 2 μM [methyl-14C]S-adenosylmethionine (AdoMet) or 2 μM [methyl-14C]methionine. Subsequent addition of phospholipase C to the suspension removed 95% of the radioactivity from phospholipids methylated by [methyl-14C]AdoMet within a few minutes, but was without effect on phospholipids methylated by [methyl-14C]methionine radioactivity from the latter could, nevertheless, be removed by phospholipase C after permeabilization of the cells with digitonin. The results clearly show that the methyl group of exogenous AdoMet, contrary to that of methionine, is transferred on to phospholipids located on the external face of the plasma membrane. Accordingly, pretreatment of isolated hepatocytes with trypsin prevented the methylation of phospholipids from exogenous AdoMet by 60-80%, whereas it was almost without effect when exogenous methionine was the methyl donor. Our data corroborate previous work [Bontemps and Van den Berghe (1997) Biochem. J. 327, 383-389], which indicated that AdoMet methylates hepatocyte phospholipids without penetrating the cells.

Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endothelial cells

2001 ◽  
Vol 356 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Frank PETRAT ◽  
Herbert de GROOT ◽  
Ursula RAUEN
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Laser Scanning ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Liver Endothelial Cells ◽  
Isolated Rat

The pool of cellular chelatable iron (‘free iron’, ‘low-molecular-weight iron’, the ‘labile iron pool’) is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8±2.6μM within the cytosol and of at least 4.8μM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6±2.9μM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3±2.6μM; nucleus, 11.8±3.9μM; mitochondria, 9.2±2.7μM); in addition, chelatable iron (approx. 16±4μM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.

scholarly journals Uncoupling effect of polyunsaturated fatty acid deficiency in isolated rat hepatocytes:effect on glycerol metabolism

1996 ◽  
Vol 317 (3) ◽  
pp. 667-674 ◽  
Author(s):  
Marie-Astrid PIQUET ◽  
Eric FONTAINE ◽  
Brigitte SIBILLE ◽  
Céline FILIPPI ◽  
Christiane KERIEL ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Dehydrogenase Activity ◽  
Potassium Cyanide ◽  
Glycerol Metabolism ◽  
Glycolytic Flux ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Isolated Rat ◽  
Deficient Group

The effects of a 4-week deficiency in polyunsaturated fatty acids (PUFA) in isolated rat hepatocytes have been investigated for oxidative phosphorylation and fatty acid, dihydroxyacetone (DHA) or glycerol metabolism. Oxygen uptake was significantly increased (by 20%) with or without fatty acid addition (octanoate or oleate) in the PUFA-deficient group compared with controls. The effect persisted after oligomycin addition but not after that of potassium cyanide, leading to the conclusion that, in these intact cells, the mitochondria were uncoupled. The PUFA-deficient group exhibited a significant decrease in the cytosolic ATP/ADP ratio, whereas the mitochondrial ratio was not affected. PUFA deficiency led to a 16% decrease in DHA metabolism owing to a 34% decrease in glycerol kinase activity; the significant decrease in the ATP/ADP ratio was accompanied by an increase in the fractional glycolytic flux. In contrast, glycerol metabolism was significantly enhanced in the PUFA-deficient group. The role of the glycerol 3-phosphate dehydrogenase step in this stimulation was evidenced in hepatocytes perifused with glycerol and octanoate in the presence of increased concentrations of 2,4-dinitrophenol (Dnp): uncoupling with Dnp led to an enhancement of glycerol metabolism, as found in PUFA deficiency, although it was more pronounced than in controls. The matrix/cytosol gradients for redox potential and ATP/ADP ratio were lower in cells from PUFA-deficient rats, suggesting a decreased mitochondrial membrane potential in accordance with the uncoupling effect. Moreover, a doubling of the mitochondrial glycerol 3-phosphate dehydrogenase activity in the PUFA-deficient group compared with controls led us to conclude that the activation of glycerol metabolism is the consequence of two mitochondrial effects: uncoupling and an increase in glycerol 3-phosphate dehydrogenase activity.

scholarly journals 4 β-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes

1986 ◽  
Vol 238 (3) ◽  
pp. 737-743 ◽  
Author(s):  
J M Staddon ◽  
R G Hansford
Keyword(s):  
Protein Kinase C ◽  
Cyclic Amp ◽  
Phosphodiesterase Inhibitor ◽  
Rat Hepatocytes ◽  
Dibutyryl Cyclic Amp ◽  
Isolated Rat Hepatocytes ◽  
Kinase C ◽  
Subsequent Addition ◽  
Isolated Rat ◽  
Sustained Increase

Hepatocytes were isolated from rats and then loaded with the fluorescent Ca2+ indicator quin2. Glucagon caused a sustained increase (at least 5 min) in the fluorescence of the quin2-loaded cells; the increase was much greater than that observed with control, non-quin2-loaded, cells. These observations indicate that glucagon caused an increase in cytoplasmic free Ca2+ concentration [(Ca2+]c). The effects of glucagon were mimicked if forskolin (to activate adenylate cyclase), dibutyryl cyclic AMP or bromo cyclic AMP were added directly to the cells. Thus an increase in cyclic AMP concentration may mediate the effect of glucagon on [Ca2+]c. If 4 beta-phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) was added to the cells before glucagon, the magnitude of the increase in [Ca2+]c was greatly diminished. If PMA was added after glucagon it caused a lowering of [Ca2+]c. These effects of PMA on the glucagon-induced increase in [Ca2+]c could not be mimicked if [Ca2+]c was increased by the Ca2+-ionophore ionomycin. Thus an event involved in the mechanism by which glucagon increases [Ca2+]c appears to be required for the action of PMA. If [Ca2+]c was increased by forskolin, dibutyryl cyclic AMP or bromo cyclic AMP, the effect of PMA on [Ca2+]c was similar to that observed when glucagon was used to elevate [Ca2+]c. When [Ca2+]c was raised by dibutyryl cyclic AMP the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent the subsequent addition of PMA from causing [Ca2+]c to decrease. These observations suggest that PMA can inhibit the cyclic AMP-induced increase in [Ca2+]c independently of any changes in cyclic AMP concentration. Glucagon appears to increase [Ca2+]c by releasing intracellular stores of Ca2+ and stimulating net influx of Ca2+ into the cell; PMA greatly diminishes both of these effects.

Effect of mitochondrial clustering on O2 supply in hepatocytes

1984 ◽  
Vol 247 (1) ◽  
pp. C83-C89 ◽  
Author(s):  
D. P. Jones
Keyword(s):  
Diffusion Coefficient ◽  
Rat Hepatocytes ◽  
Dependence Analysis ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Isolated Mitochondria ◽  
Intracellular Diffusion ◽  
O2 Supply ◽  
Intermediate Value ◽  
Isolated Rat

Isolated rat hepatocytes were treated with digitonin to selectively disrupt the plasma membrane and allow study of the O2 dependence of mitochondria within the cell cytoskeleton, but without cytosol. Half-maximal oxidation of cytochrome c occurred at 2.0 microM O2 in treated cells incubated under State 3 conditions, whereas in intact cells it was 6.0 microM and in isolated mitochondria (State 3) 0.69 microM. the intermediate value for treated cells indicates that both the geometry of mitochondrial packing and the intracellular diffusion coefficient are important in determining intracellular mitochondrial O2 dependence. Analysis of intracellular diffusion, assuming that the mitochondrial clustering increases the effective mitochondrial radius, indicates that an intracellular diffusion coefficient of between 10(-6) and 4 X 10(-6) cm2 . s-1 and an effective mitochondrial radius of approximately 2 micron would account for the observed intracellular O2 dependence of mitochondrial function.

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