Effects of anaerobiosis, glucose, insulin and glucagon on glycogen metabolism in isolated parenchymal rat liver cells

Seglen, Per Ottar

doi:10.1016/0014-5793(73)80398-9

Cited by 29 publications

(9 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3 shows the time course of uptake with 20 mM valine (extracellular concentration) under aerobic and anaerobic conditions. It can be seen that complete anoxia (100 % NZ), which stops energy-dependent processes such as gluconeogenesis 1291, glycogen formation [29,36], protein synthesis [37] and urea formation 138,391 in these cells, is without any significant effect on valine uptake. Hence, valine transport appears to be energy-independent.…”

Section: Incorporationmentioning

confidence: 99%

Valine Uptake and Incorporation into Protein in Isolated Rat Hepatocytes

Seglen

Solheim

1978

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

1. In isolated rat hepatocytes, valine is transported by a high-&system ( K , = 43mM, V = 22nmol. mg protein-' . min-I), which is non-concentrative and energy-independent. Valine transport proceeds equally well in both directions (influx and efflux) and is subject to accelerative exchange. By these properties, the transport system can be defined as a facilitated diffusion (passive transport). There is no evidence for valine uptake by simple (physical) diffusion (i.e. no uptake at 0 "C), and it is suggested that the latter takes place only in damaged or dysfunctional cells.2. Valine transport is temperature-dependent, with an activation energy of 60 kJ/mol in the temperature range 30-40 "C. Below 25 "C the activation energy increases to 103 kJ/mol, possibly reflecting a phase transition in the cell membrane. Protein synthesis and protein degradation both have an activation energy of about 100 kJ/mol throughout the temperature range 10-40 "C.3. At 37 "C, a tracer dose of [14C]valine equilibrates so rapidly across the cell membrane that it is impossible to tell whether the radioactive valine incorporated into protein is taken from an intracellular or extracellular amino acid pool. In addition, interpretation of the protein labelling pattern is complicated by endogenous protein degradation and subsequent amino acid release, which results in both isotope dilution and a gradual stimulation of protein synthesis.4. Since valine transport is temperature-dependent, a slow influx or efflux of valine can be produced by lowering the incubation temperature to the range 15-25 "C. Low temperatures also reduce the disturbing influence of protein degradation, while protein synthesis still proceeds at a sufficient rate to obtain analyzable incorporation kinetics.With only a tracer dose of [l"C]valine in the extracellular medium, the rate of protein labelling under these conditions can be seen to follow closely the changes in the intracellular rather than in the extracellular radioactivity, both during influx and efflux of ['4C]valine. However, when the extracellular valine concentration is maintained several-fold higher than the intracellular concentration, protein labelling correlates better with the extracellular than with the intracellular radioactivity. These results suggest that both the intracellular and the extracellular valine pools can directly provide precursors for protein synthesis, the relative contribution from each pool being proportional to the relative valine concentration in that pool, as suggested by Khairallah and co-workers [Biochem.

show abstract

Section: Incorporationmentioning

confidence: 99%

Valine Uptake and Incorporation into Protein in Isolated Rat Hepatocytes

Seglen

Solheim

1978

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…in stoppered tubes under a N2 atmosphere, results in the inhibition of energy-requiring cellular processes [21,53,54]. Fig.…”

Section: Efseect Of Anoxia On Protein Synthesis and Protein Degradationmentioning

confidence: 99%

Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin

Seglen

Grinde

Solheim

1979

European Journal of Biochemistry

Self Cite

456

252

View full text Add to dashboard Cite

1. Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre-labelled protein. To reduce background radioactivity, the intracellular ['4C]valine pool was depleted by serial extraction at 37 "C, effecting equilibration between the intracellular pool and the valine-free extracellular medium. After extraction, a small, non-equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine-containing oligopeptides derived from protein degradation.2. The ['4C]valine released from degraded protein was not significantly re-utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes.3. From cells pre-labelled for 24 h Ivi vivo, ['4C]valine was released linearly at a rale of 5 %/h, probably representing the true over-all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4C1) inhibited 70% of the degradation, presumably the contribution by the lysosomal pathway.From 1-h pre-labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short-lived proteins are degraded by the non-lysosomal pathway(s).4. Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect ( < 20 %) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non-lysosomal protein degradation as well.5. Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein-free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation.6. Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one-half of the degradation by the nonlysosomal pathways appears to be energy-dependent.The mechanisms responsible for the general turnover of endogeneous protein in animal cells are not well known. Lysosomes (autophagy) appear to play a major role [I -41 but in addition a numbe...

show abstract

“…As stated by Krahl (48), the primordial function of insulin consists of promoting the formation and storage of macromolecules such as glycogen, lipids, and proteins. Particular efforts were devoted to the study of the effects of insulin on glycogenesis, but contradictory results on the rapid effect of the hormone were obtained during in vivo (6,10,22,24) and in vitro investigations (35,55,64,65). In isolated hepatocytes, the early stimulation of glycogen synthetase and of the synthesis of polysaccharide by insulin has not been demon-878 THE JOURNAL OF CELL BIOLOGY 9 VOLUME 74, 1977 9 pages 878-900 strated except in a few cases (1,82).…”

mentioning

confidence: 99%

Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

Bernaert¹,

Wanson²,

Drochmans³

et al. 1977

The Journal of Cell Biology

View full text Add to dashboard Cite

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear, after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells show an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes.The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the cultures induces, after 48 and 72 h, a three-to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.Primary cultures of hepatocytes isolated from adult rats were used to study the effect of insulin on various metabolic pathways in the liver. As stated by Krahl (48), the primordial function of insulin consists of promoting the formation and storage of macromolecules such as glycogen, lipids, and proteins. Particular efforts were devoted to the study of the effects of insulin on glycogenesis, but contradictory results on the rapid effect of the hormone were obtained during in vivo (6,10,22,24) and in vitro investigations (35,55,64,65). In isolated hepatocytes, the early stimulation of glycogen synthetase and of the synthesis of polysaccharide by insulin has not been demon-

show abstract

Effects of anaerobiosis, glucose, insulin and glucagon on glycogen metabolism in isolated parenchymal rat liver cells

Cited by 29 publications

References 24 publications

Valine Uptake and Incorporation into Protein in Isolated Rat Hepatocytes

Valine Uptake and Incorporation into Protein in Isolated Rat Hepatocytes

Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin

Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

Contact Info

Product

Resources

About