scholarly journals Interactions of dietary fat and 2,5-anhydro-d-mannitol on energy metabolism in isolated rat hepatocytes

2002 ◽  
Vol 282 (3) ◽  
pp. R715-R720 ◽  
Author(s):  
Hong Ji ◽  
Grazyna Graczyk-Milbrandt ◽  
Mary D. Osbakken ◽  
Mark I. Friedman
Keyword(s):  
Oxygen Consumption ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Metabolic Effects ◽  
Atp Content ◽  
Isolated Rat Hepatocytes ◽  
Palmitate Oxidation ◽  
Low Carbohydrate ◽  
Lower Oxygen ◽  
Hepatocyte Metabolism

The fructose analog 2,5-anhydro-d-mannitol (2,5-AM) stimulates feeding in rats by reducing ATP content in the liver. These behavioral and metabolic effects occur with rats fed a high-carbohydrate/low-fat (HC/LF) diet, but they are prevented or attenuated when the animals eat high-fat/low-carbohydrate (HF/LC) food. To examine the metabolic bases for this effect of diet, we assessed the actions of 2,5-AM on ATP content, oxygen consumption, and substrate oxidation in isolated hepatocytes from rats fed one of the two diets. Compared with cells from rats fed the HC/LF diet (“HC/LF” cells), cells from rats fed the HF/LC diet (“HF/LC” cells) had similar ATP contents but lower oxygen consumption, decreased fructose, and increased palmitate oxidation. 2,5-AM did not decrease ATP content or oxygen consumption in HF/LC cells as much as it did in HC/LF hepatocytes, and it only affected fructose and palmitate oxidation in HC/LF cells.31P-NMR spectroscopy indicated that differences in phosphate trapping accounted for differences in depletion of ATP by 2,5-AM. These results suggest that intake of the HF/LC diet prevents the eating response and attenuates the decline in liver ATP by shifting hepatocyte metabolism to favor fat over carbohydrate as an energy-yielding substrate.

Phenylarsine oxide inhibition of endocytosis: effects on asialofetuin internalization

1989 ◽  
Vol 257 (2) ◽  
pp. C182-C184 ◽  
Author(s):  
A. E. Gibson ◽  
R. J. Noel ◽  
J. T. Herlihy ◽  
W. F. Ward
Keyword(s):  
Oxygen Consumption ◽  
Rat Hepatocytes ◽  
Atp Content ◽  
Phenylarsine Oxide ◽  
Careful Evaluation ◽  
Isolated Rat Hepatocytes ◽  
Energy Stores ◽  
Experimental Preparation ◽  
Oxide Inhibition ◽  
Slight Inhibition

The aim of this work was to establish to what extent the concentrations over which phenylarsine oxide (PAO) inhibits oxygen consumption and decreases cellular ATP content overlap with those used to inhibit protein internalization. The effects of PAO (1-100 microM) on 125I-labeled asialofetuin internalization, oxygen consumption, ATP content, and lactate dehydrogenase (LDH) latency of isolated rat hepatocytes were determined. Ten micromoles/liter PAO blocked 125I-asialofetuin internalization but had no effect on ATP content up to 20 min, only a slight inhibition (18%) on oxygen consumption, and no effect on LDH latency. Higher concentrations of PAO had increasingly deleterious effects on the parameters. These results show that higher concentrations of PAO severely affect the energy stores as well as integrity of the hepatocyte. We concluded that 1) use of PAO requires careful evaluation of its effects in each experimental preparation and 2) it should be possible to establish time and concentration parameters for use of PAO as an inhibitor of endocytosis while minimizing the influence of its other cellular actions.

scholarly journals The roles of different protein kinases and of calmodulin in the effects of Ca2+ mobilization on 3-hydroxy-3-methylglutaryl-CoA reductase activity in isolated rat hepatocytes

1991 ◽  
Vol 273 (2) ◽  
pp. 485-488 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Protein Kinase ◽  
Reductase Activity ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Lysosomal Proteolysis ◽  
Dependent Protein ◽  
Coa Reductase

The roles of protein kinase C, Ca2+/calmodulin-dependent protein kinase and AMP-activated protein kinase in the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase induced by Ca2(+)-mobilizing conditions in isolated hepatocytes were investigated. Only partial evidence for the involvement of AMP-activated kinase was found. Antagonism of calmodulin action prolonged the decrease in expressed/total activity ratio induced by vasopressin plus glucagon. Protease inhibitors active against Ca2(+)-dependent cytosolic proteases or lysosomal proteolysis did not attenuate the loss of total HMG-CoA reductase induced by glucagon plus vasopressin, but calmodulin antagonists largely prevented this effect.

Regulation of ANG II and AVP receptors in isolated hepatocytes of pregnant rats

1990 ◽  
Vol 258 (4) ◽  
pp. E597-E605
Author(s):  
G. Massicotte ◽  
L. Coderre ◽  
J. L. Chiasson ◽  
G. Thibault ◽  
E. L. Schiffrin ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Maximum Response ◽  
Receptor Subtype ◽  
Ang Ii ◽  
Single Class ◽  
Pregnant Rats ◽  
Isolated Rat Hepatocytes ◽  
Biological Responses

Recent evidence suggests that angiotensin II (ANG II) and vasopressin (AVP) act on the liver via specific receptors. We have examined the binding properties of these receptors in isolated rat hepatocytes and studied the regulation of the biological responses to ANG II and AVP during pregnancy in the rat. In contrast to [3H]ANG II, 125I-labeled-[Sar1-Ile8]ANG II was markedly resistant to degradation by isolated liver cells. Displacement and saturation experiments with this iodinated antagonist revealed the presence of a single class of binding sites [2 x 10(5) sites/cell, dissociation constant (KD) = 1.0 nM]. The potency of ANG II analogues to displace 125I-[Sar1-Ile8]-ANG II agrees closely with data reported for vascular smooth muscle cells. Isolated hepatocytes have approximately 8 x 10(4) [3H]AVP binding sites/cell (KD = 1.0 nM) based on saturation experiments. AVP analogues selectively displaced [3H]AVP, suggesting the presence of V1-AVP receptor subtype. The maximum response of [Sar1]ANG II-induced glycogenolysis in the cells was decreased during gestation, whereas the effective concentration producing 50% of maximum response (EC50) was significantly increased (0.15-0.28 nM) when compared with cells from nonpregnant animals. In pregnancy, receptors for 125I-[Sar1-Ile8]ANG II were not changed in affinity (KD) or in density (Bmax). The maximum response and EC50 of AVP on liver glycogenolysis were not significantly decreased during pregnancy, whereas an increased number of AVP binding sites (from 5.0 +/- 0.5 x 10(4) to 11.0 +/- 1.7 x 10(4)) with similar KD was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations

2003 ◽  
Vol 370 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Roland B. GREGORY ◽  
Gregory J. BARRITT
Keyword(s):  
High Selectivity ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Cation Channels ◽  
Isolated Rat Hepatocytes ◽  
Pharmacological Agents ◽  
Crac Channels ◽  
Kinetics Of ◽  
Ca2 Channels

Store-operated Ca2+ channels in liver cells have been shown previously to exhibit a high selectivity for Ca2+ and to have properties indistinguishable from those of Ca2+-release-activated Ca2+ (CRAC) channels in mast cells and lymphocytes [Rychkov, Brereton, Harland and Barritt (2001) Hepatology 33, 938—947]. The role of CRAC channels in the maintenance of hormone-induced oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) in isolated rat hepatocytes was investigated using several inhibitors of CRAC channels. 2-Aminoethyl diphenylborate (2-APB; 75μM), Gd3+ (1μM) and 1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365; 50μM) each inhibited vasopressin- and adrenaline (epinephrine)-induced Ca2+ oscillations (measured using fura-2). The characteristics of this inhibition were similar to those of inhibition caused by decreasing the extracellular Ca2+ concentration to zero by addition of EGTA. The effect of 2-APB was reversible. In contrast, LOE-908 {(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamidemesylate}(30μM), used commonly to block Ca2+ inflow through intracellular-messenger-activated, non-selective cation channels, did not inhibit the Ca2+ oscillations. In the absence of added extracellular Ca2+, 2-APB, Gd3+ and SK&F 96365 did not alter the kinetics of the increase in [Ca2+]cyt induced by a concentration of adrenaline or vasopressin that induces continuous Ca2+ oscillations at the physiological extracellular Ca2+ concentration. Ca2+ inflow through non-selective cation channels activated by maitotoxin could not restore Ca2+ oscillations in cells treated with 2-APB to block Ca2+ inflow through CRAC channels. Evidence for the specificity of the pharmacological agents for inhibition of CRAC channels under the conditions of the present experiments with hepatocytes is discussed. It is concluded that Ca2+ inflow through CRAC channels is required for the maintenance of hormone-induced Ca2+ oscillations in isolated hepatocytes.

Propranolol metabolism by isolated hepatocytes from normal and cirrhotic rat livers: the effect of albumin

1990 ◽  
Vol 68 (6) ◽  
pp. 657-662 ◽  
Author(s):  
Louise Gariepy ◽  
Daphna Fenyves ◽  
Jean-Luc Petit ◽  
Ginette Raymond ◽  
Jean-Pierre Villeneuve
Keyword(s):  
Free Fraction ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hepatic Uptake ◽  
Albumin Concentration ◽  
Isolated Rat Hepatocytes ◽  
Subsequent Elimination ◽  
Rat Livers ◽  
Mediated Catalysis ◽  
Isolated Rat

Several recent reports have shown that the hepatic uptake and subsequent elimination of some substrates is faster in the presence of albumin than in its absence, as if some of the substrate bound to albumin was also available for uptake. In the present study, we examined the effect of albumin on the clearance of propranolol by isolated rat hepatocyte suspensions. The clearance of total drug decreased progressively as albumin concentration increased. There was also a progressive decrease in the free fraction of propranolol and the net result was an increase in the clearance of unbound drug (+50% at 40 g/L albumin). This increase was not due to an oncotic pressure effect of albumin, nor to the presence of fatty acids bound to albumin. The clearance of propranolol by isolated hepatocytes from cirrhotic rats was decreased compared with controls (−50%), and albumin also increased propranolol free clearance, albeit to a lesser extent than in control animals. Our results indicate that albumin facilitates the elimination of propranolol by hepatocytes, possibly because of surface-mediated catalysis of the albumin–propranolol complexes.Key words: propranolol clearance, albumin, isolated rat hepatocytes, cirrhosis.

scholarly journals Effects of monensin on insulin interactions with isolated hepatocytes. Evidence for inhibition of receptor recycling and insulin degradation

1986 ◽  
Vol 234 (2) ◽  
pp. 463-468 ◽  
Author(s):  
J Whittaker ◽  
V A Hammond ◽  
R Taylor ◽  
K G M M Alberti
Keyword(s):  
Time Course ◽  
Rat Hepatocytes ◽  
Insulin Binding ◽  
Isolated Hepatocytes ◽  
Insulin Degradation ◽  
Receptor Recycling ◽  
Surface Binding ◽  
Binding Studies ◽  
Isolated Rat Hepatocytes ◽  
Receptor Concentration

Recent evidence suggests that, during endocytosis, receptors for many polypeptide ligands are spared degradation and are recycled to the plasma membrane for re-utilization. The univalent ionophore monensin was shown to inhibit membrane recycling. We therefore examined its effects on insulin interactions with isolated rat hepatocytes to characterize further receptor endocytosis and recycling in these cells. At 10 degrees C, in the absence of endocytosis, no change in insulin binding was observed. However, at 37 degrees C a concentration-dependent decrease in 125I-insulin binding was seen in the presence of insulin; this reached a maximum of 60% at 1 nM-insulin. Competitive binding studies showed this to be due to a 50-60% decrease in cell-surface insulin-receptor concentration, although the total cellular receptor concentration remained unchanged, suggesting that monensin causes the intracellular sequestration of receptors. Time-course studies of the processing of 2.5 nM-insulin showed that monensin produced a 50-60% decrease in surface binding, accompanied by a similar decrease in internalization and total inhibition of insulin degradation. When hepatocytes with 125I-insulin prebound to their surface receptors at 10 degrees C were warmed to 37 degrees C, monensin had no effect on internalization, but caused marked impairment of intracellular insulin degradation. It is concluded that monensin inhibits receptor recycling and cellular insulin degradation.

Uptake of unconjugated bilirubin by isolated rat hepatocytes

1979 ◽  
Vol 236 (1) ◽  
pp. C9-C14 ◽  
Author(s):  
T. Iga ◽  
D. L. Eaton ◽  
C. D. Klaassen
Keyword(s):  
Lipid Membrane ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hepatic Uptake ◽  
Unconjugated Bilirubin ◽  
Metabolic Inhibitors ◽  
Isolated Rat Hepatocytes ◽  
Intracellular Binding ◽  
High Degree ◽  
Isolated Rat

The mechanism responsible for the hepatic uptake of unconjugated bilirubin was examined in isolated rat hepatocytes from control and phenobartital-pretreated rats. The uptake was extremely rapid and the equilibrium between cell and medium was attained within 60 s with a 100-fold higher concentration in the cell than the medium. The initial velocity of uptake (Vo) exhibited a linear relationship to the bilirubin concentration in the medium. Pretreatment of cells with various metabolic inhibitors had no effect on the uptake of unconjugated bilirubin. Ouabain did significantly decrease Vo, but replacement of sodium ion with choline or lithium had no effect on bilirubin uptake. The organic acids sulfobromophthalein (112 muM) and taurocholic acid (50 (muM) and two steroidal compounds, diethylstilbestrol (50 muM) and spironolactone (50 muM), had no effect on the uptake of bilirubin. It is suggested that bilirubin gains access to the hepatocyte interior by passive diffusion into and through the lipid membrane and that intracellular binding may explain the high degree of bilirubin accumulation associated with the isolated hepatocytes.

scholarly journals Sugar uptake by fluid-phase pinocytosis and diffusion in isolated rat hepatocytes

1987 ◽  
Vol 243 (3) ◽  
pp. 655-660 ◽  
Author(s):  
P B Gordon ◽  
H Høyvik ◽  
P O Seglen
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Free Sugar ◽  
Sugar Uptake ◽  
Isolated Rat Hepatocytes ◽  
Fluid Phase Endocytosis ◽  
And Diffusion ◽  
Isolated Rat

Measurements of sugar pinocytosis (fluid-phase endocytosis of radiolabelled sucrose, lactose and raffinose) in freshly isolated rat hepatocytes are disturbed by sugar diffusing into the cells through plasma-membrane blebs. Non-pinocytic entry may be even more pronounced at 0 degrees C, and is a major contributor to ‘background’ radioactivity. By electrodisruption of the plasma membrane, a distinction can be made between pinocytotically sequestered sugar and free sugar that has entered the cytosol by diffusion. Pinocytosis proceeds at a rate of 2%/h (relative to the intracellular fluid volume), whereas the rate of sucrose entry by diffusion is more than twice as high. Three pinocytotic compartments are distinguishable in isolated hepatocytes: (1) a rapidly recycling compartment, which is completely destroyed by electrodisruption, and which may represent pinocytic channels continuous with the plasma membrane; (2) a non-recycling (or very slowly recycling) electrodisruption-resistant compartment, which allows accumulation of the lysosomally hydrolysable sugar lactose, and which therefore must represent non-lysosomal vacuoles (endosomes?); (3) a lysosomal compartment (non-recycling, electrodisruption-resistant), which accumulates raffinose and sucrose, but which hydrolyses lactose. The last two compartments can be partially resolved in metrizamide/sucrose density gradients by the use of different sugar probes.

scholarly journals Characterization of the liver P2-purinoceptor involved in the activation of glycogen phosphorylase

1986 ◽  
Vol 240 (2) ◽  
pp. 367-371 ◽  
Author(s):  
S Keppens ◽  
H De Wulf
Keyword(s):  
Binding Sites ◽  
Glycogen Phosphorylase ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Rank Order ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
P2 Purinoceptors

Evidence has been presented for the existence in rat liver of P2-purinoceptors which are involved in the control of glycogenolysis. Isolated rat hepatocytes and purified liver plasma membranes have been used to study the binding of the ATP analogue adenosine 5′-[alpha- [35S]thio]triphosphate (ATP alpha [35S]) to these postulated P2-purinoceptors. The nucleotide analogue behaves as a full agonist for the activation of glycogen phosphorylase in isolated hepatocytes, 0.3 microM being required for half-maximal activation. Specific binding of ATP alpha [35S] to hepatocytes and plasma membranes occurs within 1 min and is essentially reversible. The analysis of the dose-dependency at equilibrium indicates the presence of binding sites with Kd of 0.23 microM with hepatocytes and Kd of 0.11 microM with plasma membranes. The relative affinities of 10 nucleotide analogues were deduced from competition experiments for ATP alpha [35S] binding to hepatocytes, and these correlated highly with their biological activity (activation of glycogen phosphorylase in hepatocytes). For all the agonists, binding occurs in the same concentration range as the biological effect. These data clearly suggest that the detected binding sites correspond to the physiological P2-purinoceptors involved in the regulation of liver glycogenolysis. The rank order of potency of some ATP analogues suggests that liver possesses the P2Y-subclass of P2-purinoceptors.

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