Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes

We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also examined. The following observations were made: (1) with glucose alone, net glycogen production was low. Inhibition of glucose-6-phosphate translocase increased intracellular glucose 6-phosphate (3-fold), glycogen accumulation (5-fold) without change in active (dephosphorylated) glycogen synthase (GSa) activity, and lactate production (4-fold). With both glucose 6-phosphate translocase and glycogen phosphorylase inhibited, glycogen deposition increased 8-fold and approached reported in vivo rates of glycogen deposition during the fasted → fed transition. Addition of a physiological mixture of amino acids in the presence of glucose increased glycogen accumulation (4-fold) through activation of GS and inhibition of glucose-6-phosphatase flux. Addition of oleate with glucose present decreased glycolytic flux and increased the flux through glucose 6-phosphatase with no change in glycogen deposition. With glucose 6-phosphate translocase inhibited by S4048, oleate increased intracellular glucose 6-phosphate (3-fold) and net glycogen production (1.5-fold), without a major change in GSa activity. It is concluded that glucose cycling in hepatocytes prevents the net accumulation of glycogen from glucose. Amino acids activate GS and inhibit flux through glucose-6-phosphatase, while oleate inhibits glycolysis and stimulates glucose-6-phosphatase flux. Variation in glucose 6-phosphate does not always result in activity changes of GSa. Activation of glucose 6-phosphatase flux by fatty acids may contribute to the increased hepatic glucose production as seen in Type 2 diabetes.

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Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes

Biochemical Journal ◽

10.1042/bj2480429 ◽

1987 ◽

Vol 248 (2) ◽

pp. 429-437 ◽

Cited By ~ 68

Author(s):

A Lavoinne ◽

A Baquet ◽

L Hue

Keyword(s):

Glycogen Phosphorylase ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Lactate Production ◽

Rat Hepatocytes ◽

Diabetic Rats ◽

Purine Analogues ◽

Isolated Rat Hepatocytes ◽

Body Production ◽

Stimulation Of

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.

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Nitric oxide inhibits glycogen synthesis in isolated rat hepatocytes

Biochemical Journal ◽

10.1042/bj3301045 ◽

1998 ◽

Vol 330 (2) ◽

pp. 1045-1049 ◽

Cited By ~ 50

Author(s):

Fleur SPRANGERS ◽

P. Hans SAUERWEIN ◽

A. Johannes ROMIJN ◽

M. George van WOERKOM ◽

J. Alfred MEIJER

Keyword(s):

Nitric Oxide ◽

Glucose Metabolism ◽

Phosphoenolpyruvate Carboxykinase ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Rat Hepatocytes ◽

Inhibitory Effect ◽

No Donor ◽

Isolated Rat Hepatocytes ◽

Intracellular Glucose

There is increasing evidence for the existence of intrahepatic regulation of glucose metabolism by Kupffer cell products. Nitric oxide (NO) is known to inhibit gluconeogenic flux through pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, NO may also influence glucose metabolism at other levels. Using hepatocytes from fasted rats incubated with the NO-donor S-nitroso-N-acetylpenicillamine, we have now found that the synthesis of glycogen from glucose is even more sensitive to inhibition by NO than gluconeogenesis. Inhibition of glycogen production by NO was accompanied by a rise in intracellular glucose 6-phosphate and UDPglucose. Activity of glycogen synthase, as measured in extracts of hepatocytes after the cells had been exposed to NO, was decreased. Experiments with gel-filtered liver extracts revealed that inhibition of glycogen synthase was caused by an inhibitory effect of NO on the conversion of glycogen synthase b into glycogen synthase a.

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Molybdate and tungstate act like vanadate on glucose metabolism in isolated hepatocytes

Biochemical Journal ◽

10.1042/bj2820659 ◽

1992 ◽

Vol 282 (3) ◽

pp. 659-663 ◽

Cited By ~ 37

Author(s):

C Fillat ◽

J E Rodríguez-Gil ◽

J J Guinovart

Keyword(s):

Glucose Metabolism ◽

Glycogen Phosphorylase ◽

Kinase Activity ◽

Glycogen Synthase ◽

Rat Hepatocytes ◽

Isolated Hepatocytes ◽

The Other ◽

Co2 Production ◽

Glycolytic Flux ◽

Dependent Mechanism

In rat hepatocytes, molybdate and tungstate inactivate glycogen synthase by a mechanism independent of Ca2+ and activate glycogen phosphorylase by a Ca(2+)-dependent mechanism. On the other hand, both molybdate and tungstate increase fructose 2,6-bisphosphate levels and counteract the decrease in this metabolite induced by glucagon. These effectors do not directly modify 6-phosphofructo-2-kinase activity, even though they partially counteract the inactivation of this enzyme induced by glucagon. These effects are related to an increase on the glycolytic flux, as indicated by the increase in L-lactate and CO2 production and the decrease in glucose 6-phosphate levels in the presence of glucose. All these effects are similar to those previously reported for vanadate, although molybdate and tungstate are less effective than vanadate. These results could indicate that molybdate, tungstate and vanadate act on glucose metabolism in isolated hepatocytes by a similar mechanism of action.

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Control of gluconeogenesis and of enzymes of glycogen metabolism in isolated rat hepatocytes. A parallel study of the effect of phenylephrine and of glucagon

Biochemical Journal ◽

10.1042/bj1760791 ◽

1978 ◽

Vol 176 (3) ◽

pp. 791-797 ◽

Cited By ~ 69

Author(s):

Louis Hue ◽

Juan Emilio Felíu ◽

Henri-Géry Hers

Keyword(s):

Protein Kinase ◽

Pyruvate Kinase ◽

Incubation Medium ◽

Glycogen Phosphorylase ◽

Glycogen Synthase ◽

Rat Hepatocytes ◽

Glycogen Metabolism ◽

Parallel Study ◽

Isolated Rat Hepatocytes ◽

Isolated Rat

Hepatocytes isolated from the livers of fed rats were used for a comparative study of the effects of phenylephrine, vasopressin and glucagon on gluconeogenesis and on enzymes of glycogen metabolism. When hepatocytes were incubated in the presence of Ca2+, phenylephrine stimulated gluconeogenesis from pyruvate less than did glucagon, but, in contrast with this hormone, it did not affect the activities of protein kinase and pyruvate kinase, nor the concentration of phosphoenolpyruvate, and it did not decrease the release of 3H2O from [6-3H]glucose. The effects of vasopressin were similar to those of phenylephrine. Gluconeogenesis from fructose was also stimulated by phenylephrine and, more markedly, by glucagon at the expense of the conversion of fructose into lactate. Insulin was able to antagonize the stimulatory effect of phenylephrine on gluconeogenesis from pyruvate. When Ca2+ was removed from the incubation medium, phenylephrine still stimulated gluconeogenesis from pyruvate, but it also caused an activation of protein kinase and an inactivation of pyruvate kinase; accordingly, the concentration of phosphoenolpyruvate was increased, and, in contrast, vasopressin had no effect on all these parameters. The property of phenylephrine to cause the activation of glycogen phosphorylase was decreased by glucose or by the absence of Ca2+; it was abolished when these two conditions were combined. Glycogen synthase was inactivated by phenylephrine in the presence or the absence of Ca2+, although presumably by different mechanisms.

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Difference in glucose sensitivity of liver glycolysis and glycogen synthesis. Relationship between lactate production and fructose 2,6-bisphosphate concentration

Biochemical Journal ◽

10.1042/bj2240779 ◽

1984 ◽

Vol 224 (3) ◽

pp. 779-786 ◽

Cited By ~ 35

Author(s):

L Hue ◽

F Sobrino ◽

L Bosca

Keyword(s):

Glycogen Synthase ◽

Glycogen Synthesis ◽

Lactate Production ◽

Rat Hepatocytes ◽

Isolated Rat Hepatocytes ◽

Cytosolic Proteins ◽

Glucose Injection ◽

Opposite Situation ◽

Glucose Sensitivity ◽

Fructose 1,6 Bisphosphate

Incubation of isolated rat hepatocytes from fasted rats with 0-6 mM-glucose caused an increase in [fructose 2,6-bisphosphate] (0.2 to about 5 nmol/g) without net lactate production. A release of 3H2O from [3-3H]glucose was, however, detectable, indicating that phosphofructokinase was active and that cycling occurred between fructose 6-phosphate and fructose 1,6-bisphosphate. A relationship between [fructose 2,6-bisphosphate] and lactate production was observed when hepatocytes were incubated with [glucose] greater than 6 mM. Incubation with glucose caused a dose-dependent increase in [hexose 6-phosphates]. The maximal capacity of liver cytosolic proteins to bind fructose 2,6-bisphosphate was 15 nmol/g, with affinity constants of 5 × 10(6) and 0.5 × 10(6) M-1. One can calculate that, at 5 microM, more than 90% of fructose 2,6-bisphosphate is bound to cytosolic proteins. In livers of non-anaesthetized fasted mice, the activation of glycogen synthase was more sensitive to glucose injection than was the increase in [fructose 2,6-bisphosphate], whereas the opposite situation was observed in livers of fed mice. Glucose injection caused no change in the activity of liver phosphofructokinase-2 and decreased the [hexose 6-phosphates] in livers of fed mice.

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Glycogen synthesis by rat hepatocytes

Biochemical Journal ◽

10.1042/bj1800389 ◽

1979 ◽

Vol 180 (2) ◽

pp. 389-402 ◽

Cited By ~ 119

Author(s):

J Katz ◽

S Golden ◽

P A Wals

Keyword(s):

Glycogen Phosphorylase ◽

Glycogen Content ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Rat Hepatocytes ◽

Phosphorylated Form ◽

Cell Pellet ◽

Glycogen Deposition ◽

Gluconeogenic Substrates ◽

Total Enzyme

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12–15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50–60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.

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Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1984.tb08315.x ◽

1984 ◽

Vol 142 (3) ◽

pp. 511-520 ◽

Cited By ~ 43

Author(s):

Carlos CIUDAD ◽

Marcella CAMICI ◽

Zafeer AHMAD ◽

Yuhuan WANG ◽

Anna A. DePAOLI-ROACH ◽

...

Keyword(s):

Glycogen Synthase ◽

Rat Hepatocytes ◽

Isolated Rat Hepatocytes ◽

Isolated Rat

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Effect of amino acid deprivation on initiation of protein synthesis in rat hepatocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1989.256.1.c18 ◽

1989 ◽

Vol 256 (1) ◽

pp. C18-C27 ◽

Cited By ~ 54

Author(s):

W. V. Everson ◽

K. E. Flaim ◽

D. M. Susco ◽

S. R. Kimball ◽

L. S. Jefferson

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Rat Hepatocytes ◽

Initiation Factor ◽

Synthetic Activity ◽

Perfused Liver ◽

Amino Acid Deprivation ◽

Isolated Rat Hepatocytes ◽

Reticulocyte Lysate

Conditions were defined for maintaining optimal protein synthetic activity in suspensions of freshly isolated rat hepatocytes. Under these conditions, isolated hepatocytes exhibited rates of protein synthesis and levels of polysomal aggregation equivalent to those observed in vivo and in perfused liver. Deprivation of total amino acids or single, essential amino acids resulted in a rapid decrease in the rate of protein synthesis, which was readily reversed by readdition of the deficient amino acid(s). The decrease was accompanied by a disaggregation of polysomes and an inhibition of 43S initiation complex formation, which was indicative of a limitation in the rate of initiation of protein synthesis. Extracts prepared from perfused liver deprived of amino acids were inhibitory to initiation of protein synthesis in reticulocyte lysate. The inhibition in reticulocyte lysate was accompanied by an increase in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2), suggesting activation of an eIF-2 alpha kinase or inhibition of a phosphatase in amino acid-deprived hepatocytes. This suggestion was confirmed by prelabeling hepatocytes with 32Pi before amino acid deprivation. Incorporation of 32Pi into eIF-2 alpha was two- to threefold higher in lysine-deprived cells than in hepatocytes incubated in fully supplemented medium. Overall, the results indicated that an increase in eIF-2 alpha phosphorylation was responsible for the defect in initiation of protein synthesis caused by amino acid deprivation.

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