scholarly journals Effects of fructose concentration on carbohydrate metabolism, heat production and substrate cycling in isolated rat hepatocytes

1979 ◽  
Vol 184 (3) ◽  
pp. 501-507 ◽  
Author(s):  
Dallas G. Clark ◽  
Owen H. Filsell ◽  
David L. Topping
Keyword(s):  
Heat Production ◽  
Rat Hepatocytes ◽  
Fold Increase ◽  
Hepatic Metabolism ◽  
Heat Output ◽  
O2 Consumption ◽  
O2 Uptake ◽  
Isolated Rat Hepatocytes ◽  
High Concentrations ◽  
Fructose 1,6 Bisphosphate

1. Hepatocytes from starved rats were incubated with 5mm-glucose, labelled uniformly with 14C and specifically with 3H at positions 1, 2, 3 or 6, and with fructose at concentrations of 2.5, 7.5 or 25mm. 2. In the absence of other substrates only 1% of the radioactivity initially present in [U-14C]glucose appeared in the metabolic products, CO2, lactate, pyruvate, amino acids and glycogen. 3. Fructose at 2.5mm caused a 30% increase in the glucose concentration and a 4-fold increase in the apparent oxidation of [U-14C]-glucose. 4. The formation of 3H2O from [1-3H]-, [2-3H]-, [3-3H]- or [6-3H]-glucose was 2.4, 4.3, 2.15 or 1.6% respectively in the control incubations and 4.1, 10.4, 7.7 or 5.1% with 2.5mm-fructose. 5. Fructose at 7.5 and 25mm decreased the 3H2O yields to less than the control values, but had no apparent effect on the amount of [U-14C]glucose metabolized. 6. In the incubations with 5mm-glucose and 25mm-fructose there were significant decreases in heat production, O2 consumption and in the ratio of O2 uptake to heat output. 7. Fructose at 2.5mm caused a 64% increase in heat output, but only a 43% increase in O2 uptake. 8. The radioisotopic and calorimetric data demonstrate that physiological concentrations of fructose greatly increase metabolism in hepatocytes from starved rats. These data also indicate increased cycling at glucose/glucose 6-phosphate and at fructose 6-phosphate/fructose 1,6-bisphosphate in the presence of 2.5mm-fructose, although the rates of cycling were actually decreased relative to the amount of glucose catabolized. 9. At concentrations of 2.5, 7.5 and 25mm, fructose depressed hepatocyte ATP concentrations by 20, 65 and 80% respectively. Although fructose at 7.5 and 25mm increased glucose and lactate release, O2 consumption, production of heat and formation of3H2O from [1-3H]-, [2-3H]-, [3-3H]- or [6-3H]-glucose were lowered to values equal to, or less than, controls. These effects probably reflect a severe derangement of hepatic metabolism due to excess phosphorylation of fructose when present at high concentrations.

scholarly journals Microcalorimetric measurements of heat production in brown adipocytes from control and cafeteria-fed rats

1986 ◽  
Vol 235 (2) ◽  
pp. 337-342 ◽  
Author(s):  
D G Clark ◽  
M Brinkman ◽  
S D Neville
Keyword(s):  
Oleic Acid ◽  
Heat Production ◽  
Oxidative Metabolism ◽  
Heat Output ◽  
O2 Consumption ◽  
O2 Uptake ◽  
Average Volume ◽  
Brown Adipocytes ◽  
Chemical Agents ◽  
Sequential Addition

The effects of the sequential addition of glucose, noradrenaline, propranolol and oleic acid on the rates of O2 consumption and heat production by isolated interscapular brown adipocytes from control and cafeteria-fed rats were compared. Although the chemical agents produced very similar changes in oxidative metabolism, the actual rates of O2 uptake and heat output in adipocytes from the cafeteria-fed rats, when expressed per g dry wt. of cells, were approx. 65% less than those obtained with cells from the control rats. However, when the same results were expressed per 10(8) multiloccular brown adipocytes, rather than gravimetrically, rates of O2 consumption and heat production were equivalent. Further interpretation of these data is complicated, because the average volume of multiloccular brown adipocytes from cafeteria-fed rats was 2.5 times that for multiloccular cells from control animals.

scholarly journals Inhibition of hormonal induction of tyrosine aminotransferase by polyamines in freshly isolated rat hepatocytes

1983 ◽  
Vol 214 (3) ◽  
pp. 679-685 ◽  
Author(s):  
P Auberger ◽  
M Samson ◽  
A Le Cam
Keyword(s):  
Enzyme Activity ◽  
Rat Hepatocytes ◽  
Fold Increase ◽  
Dose Dependence ◽  
Tyrosine Aminotransferase ◽  
Enzyme Inactivation ◽  
Post Translational Modification ◽  
Isolated Rat Hepatocytes ◽  
Hormonal Induction ◽  
High Concentrations

We have analysed the effects of natural aliphatic polyamines on hormonal induction of tyrosine aminotransferase (TAT) in suspensions of hepatocytes isolated from adult fed rats. Glucagon or cyclic AMP derivatives (dibutyryl and 8-bromo) used alone caused a 4-5 fold increase in enzyme activity within 4h. This effect was independent of glucocorticoids, which also increased TAT activity (2.5-fold); when combined, the effects of the two inducers were additive. Spermine and putrescine totally inhibited the hormonally-mediated increase in enzyme activity when added at the onset of incubation with the inducers. Furthermore, polyamines could block the hormonal effect at any time during the course of TAT induction, with, however, a 30 min lag period, suggesting that they must enter the cells. Hepatocytes were indeed shown to take up spermine. At low external concentrations (less than 50 microM), an Na+-dependent, saturable and concentrative mechanism was predominant; at high concentrations (greater than 0.5 mM) transport occurred mainly through a non-saturable, Na+-independent mechanism, building up intracellular concentrations slightly lower than those in the medium. Dose-dependence analysis of the polyamine effect on enzyme induction indicated that half-maximal and maximal inhibition occurred with 0.75 mM- and 2.5 mM-spermine respectively, whereas 2.5mM- and 7.5 mM-putrescine were required respectively to obtain similar effects. Spermidine was much less effective and cadaverine had virtually no effect. None of the polyamines affected the rate of decay of TAT, nor did they directly or indirectly cause enzyme inactivation, indicating that a post-translational modification was unlikely to account for the polyamine effects. Similarly, these effects could not be ascribed to a non-specific inhibition of overall protein synthesis. We conclude that, in hepatocytes, polyamines (or their metabolites) directly interfere with one or several steps controlled by hormones in the synthesis of tyrosine aminotransferase.

scholarly journals The application of microcalorimetry to the assessment of metabolic efficiency in isolated rat hepatocytes

1979 ◽  
Vol 180 (3) ◽  
pp. 631-638 ◽  
Author(s):  
Ivan G. Jarrett ◽  
Dallas G. Clark ◽  
Owen H. Filsell ◽  
John W. Harvey ◽  
Michael G. Clark
Keyword(s):  
Liver Cell ◽  
Physiological Saline ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Heat Output ◽  
Metabolic Efficiency ◽  
O2 Uptake ◽  
Isolated Rat Hepatocytes ◽  
Significant Difference ◽  
Isolated Rat

1. Heat output by suspensions of isolated rat hepatocytes was determined by using a modified batch-type microcalorimeter. 2. The ratio of O2 uptake (determined polarographically) to heat output was used to assess the metabolic efficiency of isolated hepatocytes. 3. Cells from starved or fed rats incubated in either bicarbonate-buffered physiological saline containing gelatin, or bicarbonate-buffered physiological saline containing amino acids, serum albumin and glucose showed no significant difference with respect to the ratio of O2 uptake to heat output. 4. For liver cells from 24h-starved rats, the addition of 10mm-dihydroxyacetone and 2.5mm-fructose significantly decreased the ratio of O2 uptake to heat output from 1.94±0.05 in the controls to 1.52±0.04 and 1.54±0.01μmol/J respectively. 5. Glucagon (1μm), which slightly increased both O2 uptake and heat output, did not significantly alter the ratio. 6. The addition of extracellular 10mm-NH4Cl and urease to provide an energetically wasteful cycle by ensuring hydrolysis of newly synthesized urea, lowered the ratio of O2 uptake to heat output from 1.81±0.08 to 1.47±0.06μmol/J, indicating a reduced metabolic efficiency. 7. Metabolic efficiency in rats of different dietary regimen, age and genetically based obesity was also assessed. No differences in the ratio of O2 uptake to heat output were found between liver cell suspensions prepared from rats maintained on colony diet and high-fat diet or sucrose-rich diet nor between animals ranging from 38 to 179 days of age. Comparison of the ratio of liver cell O2 uptake to heat output between homozygote Zucker fa/fa obese rats and their lean littermates showed no significant difference. 8. It is concluded that the ratio of O2 uptake to heat output for isolated hepatocytes is relatively constant unless perturbed by conditions that markedly enhance substrate cycling.

Fructose 1,6-bisphosphate protects against D-galactosamine toxicity in isolated rat hepatocytes

1994 ◽  
Vol 266 (6) ◽  
pp. C1722-C1728 ◽  
Author(s):  
T. Roig ◽  
J. R. De Oliveira ◽  
R. Bartrons ◽  
J. Bermudez
Keyword(s):  
Cell Viability ◽  
Heat Dissipation ◽  
Rat Hepatocytes ◽  
Exothermic Peak ◽  
Heat Output ◽  
Metabolic Efficiency ◽  
Isolated Rat Hepatocytes ◽  
Hepatotoxic Effect ◽  
Output Ratio ◽  
Fructose 1,6 Bisphosphate

Incubation of hepatocytes with D-galactosamine (GalN) produced a dose-dependent alteration in cell viability and a fall in ATP and fructose 2,6-bisphosphate (Fru-2,6-P2) levels. The reduction in Fru-2,6-P2 can be explained by changes in the substrates or modulators of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, because neither the adenosine 3',5'-cyclic monophosphate level nor the activity ratio of the enzyme was modified. Microcalorimetric measurements showed that GalN produced an exothermic peak followed by a progressive decrease in heat dissipation. Simultaneous administration of GalN and fructose 1,6-bisphosphate (Fru-1,6-P2) significantly increased cell viability, and concentrations of ATP and Fru-2,6-P2 and led to stable heat production. In the presence of Fru-1,6-P2 alone, hepatocytes kept ATP and Fru-2,6-P2 levels constant, whereas they increased the oxygen uptake-to-heat output ratio. Our results suggest that GalN initiates the hepatotoxic effect by means of an energy-dissipating interaction, produced before its metabolism and presumably at the membrane level, whereas Fru-1,6-P2 protects the cells against this injury in a way that prevents the initial interaction and increases the metabolic efficiency of the cell.

scholarly journals Fluorescence-activated sorting of rat hepatocytes based on their mixed function oxidase activities towards diethoxyfluorescein

1987 ◽  
Vol 247 (1) ◽  
pp. 23-28 ◽  
Author(s):  
I N H White ◽  
M L Green ◽  
R F Legg
Keyword(s):  
Dna Synthesis ◽  
Trypan Blue ◽  
Rat Hepatocytes ◽  
Fold Increase ◽  
Mixed Function Oxidase ◽  
Unscheduled Dna Synthesis ◽  
Rich Region ◽  
Trypan Blue Exclusion ◽  
Isolated Rat Hepatocytes ◽  
Viable Cells

The formation of ethoxyfluorescein and fluorescein from diethoxyfluorescein by isolated rat hepatocytes has been used as a basis for separating such cells dependent on their mixed function oxidase activities by fluorescence-activated flow cytometry. Five equal fractions defined by computer-generated regions were isolated. Non-viable cells with low fluorescence (region 1) represented 10-15% of the population, while the remainder with higher mixed function oxidase activities (regions 2-5), were greater than 95% viable by Trypan Blue exclusion. In region 1, 30% of the viable cells were binucleate, 67% diploid while in region 5, 13% were binucleate and 69% tetraploid. At 3 h after sorting, following attachment to glass coverslips, exposure of cells to methyl methanesulphonate, retrorsine or norethindrone resulted in unscheduled DNA synthesis which was 2-fold higher in the tetraploid-rich region 5, while aflatoxin B1, benzo[a]pyrene or 2-acetylaminofluorene caused a 5-fold increase in unscheduled DNA synthesis in these cells, relative to the diploid-rich hepatocytes in region 2.

Oxygen conformance of cellular respiration in hepatocytes

1993 ◽  
Vol 265 (4) ◽  
pp. L395-L402 ◽  
Author(s):  
P. T. Schumacker ◽  
N. Chandel ◽  
A. G. Agusti
Keyword(s):  
Metabolic Activity ◽  
Energy Demand ◽  
Lactic Acid Production ◽  
Rat Hepatocytes ◽  
Cellular Respiration ◽  
O2 Uptake ◽  
Rapid Reduction ◽  
Isolated Rat Hepatocytes ◽  
Cell Densities ◽  
O2 Supply

Cellular respiratory rates are normally determined by metabolic activity, but become rate limited by O2 availability if the cell O2 tension (PO2) falls below a critical value (typically 1–10 Torr). An ability to reduce metabolic activity and energy demand in response to a falling O2 availability might confer an increased resistance to a diminished O2 supply. Isolated rat hepatocytes were studied in primary culture under controlled O2 tensions. Cells were obtained by collagenase digestion and seeded into nutritive media in control and experimental spinner flasks at identical cell densities. Cells subjected to rapid reduction in PO2 (100⇢0 Torr over < 40 min) exhibited undiminished O2 uptake until PO2 fell below 10 Torr. By contrast, when cell PO2 was reduced over several hours, significant decreases in O2 uptake became evident at O2 tensions as high as 70 Torr. These decreases were associated with a reduction in ATP concentration and an increase in NAD(P)H, compared with rapidly deoxygenated cells at the same PO2. No loss in cell viability was detected after 24 h at reduced PO2. The decrease in respiratory rate was associated with an increased rate of lactic acid production relative to normoxic controls. Restoration of normoxia was associated with an immediate return of O2 uptake to control levels. These results demonstrate that hepatocytes are capable of reversibly decreasing metabolic activity and O2 demand during sustained moderate reductions in PO2, via a mechanism that appears to involve an inhibition of mitochondrial function other than O2 supply limitation. This response may alter cellular susceptibility to physiological stresses including hypoxia.

scholarly journals Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump

1987 ◽  
Vol 248 (3) ◽  
pp. 807-813 ◽  
Author(s):  
C J Lynch ◽  
S B Bocckino ◽  
P F Blackmore ◽  
J H Exton
Keyword(s):  
Cyclic Amp ◽  
Rat Hepatocytes ◽  
Previous Exposure ◽  
High Concentration ◽  
Isolated Rat Hepatocytes ◽  
Pump Activity ◽  
Heterologous Desensitization ◽  
Tumour Promoters ◽  
High Concentrations ◽  
Stimulation Of

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.

scholarly journals Stimulatory effect of the intestinal peptide PHI on glycogenolysis and gluconeogenesis in isolated rat hepatocytes

1983 ◽  
Vol 214 (3) ◽  
pp. 999-1002 ◽  
Author(s):  
J E Felíu ◽  
J Marco
Keyword(s):  
Cyclic Amp ◽  
Pyruvate Kinase ◽  
Glycogen Phosphorylase ◽  
Rat Hepatocytes ◽  
Fold Increase ◽  
Isolated Rat Hepatocytes ◽  
Mediated Effects ◽  
Dose Dependent ◽  
Isolated Rat ◽  
Stimulation Of

The newly isolated peptide PHI provoked a dose-dependent stimulation of glycogenolysis and gluconeogenesis in isolated rat hepatocytes; at 1 microM-PHI, both processes were increased 1.6-fold as compared with basal values. These PHI-mediated effects were accompanied by the activation of glycogen phosphorylase and the inactivation of pyruvate kinase. PHI (1 microM) also caused a 2-fold increase in hepatocyte cyclic AMP.

scholarly journals Utilization of endogenous and exogenous sources of substrate for cholesterol biosynthesis by isolated hepatocytes

1979 ◽  
Vol 177 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Geoffrey F. Gibbons ◽  
Clive R. Pullinger
Keyword(s):  
Cholesterol Biosynthesis ◽  
Total Sterol ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Sterol Synthesis ◽  
Isolated Rat Hepatocytes ◽  
Intermediary Metabolites ◽  
Exogenous Glucose ◽  
High Concentrations ◽  
Cholesterol Precursor

The rates of cholesterol biosynthesis in isolated rat hepatocytes were determined by using a method based on measurement of the rate of formation of desmosterol (cholesta-5,24-dien-3β-ol), which accumulates during inhibition of cholesterogenesis by the drug triparanol. Incubation of cells from normal or 24h-starved animals in a medium containing albumin, glucose, amino acids and acetate as the only organic constituents led to an accelerating rate of sterol formation during the earlier stages of a 6h incubation period. The contribution of exogenously added acetate (initial concentration 3.34mm) to sterol synthesis in both types of cells reached an early maximum and then continually declined. Exogenously added pyruvate and lactate were more efficient sources of sterol carbon than was acetate. Exogenous glucose even at relatively high concentrations (11.1mm) was incapable of providing more than 6% of the total sterol carbon. Although the proportion of total sterol carbon supplied from exogenous acetate increased with increasing concentrations of the extracellular substrate, the rates of total sterol synthesis in both types of cell remained unchanged. Similar observations were made when lactate or pyruvate was the cholesterogenic precursor in normal cells. These studies suggest that, although exogenous substrates were capable of expanding an intracellular pool of cholesterol precursor, the normal supply of intermediary metabolites was not rate-limiting for cholesterogenesis.

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