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A J Meijer

Publications and source records attributed to A J Meijer.

At least 37 records · Page 2Linked to original sources
PubMed1997

The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes.

Recent studies indicate that phosphatidylinositol 3-kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3-kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenylchromone (LY294002). The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50 30 nM) and LY294002 (IC50 10 microM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids. 3-Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3-kinase, thus providing a target for its action. It is proposed that phosphatidylinositol 3-kinase activity is required for autophagy. 3-Methyladenine inhibits autophagy by inhibition of this enzyme.

Adenine↗
PubMed1997

Autophagic proteolysis: control and specificity.

The rate of proteolysis is an important determinant of the intracellular protein content. Part of the degradation of intracellular proteins occurs in the lysosomes and is mediated by macroautophagy. In liver, macroautophagy is very active and almost completely accounts for starvation-induced proteolysis. Factors inhibiting this process include amino acids, cell swelling and insulin. In the mechanisms controlling macroautophagy, protein phosphorylation plays an important role. Activation of a signal transduction pathway, ultimately leading to phosphorylation of ribosomal protein S6, accompanies inhibition of macroautophagy. Components of this pathway may include a heterotrimeric Gi3-protein, phosphatidylinositol 3-kinase and p70S6 kinase. Recent evidence indicates that lysosomal protein degradation can be selective and occurs via ubiquitin-dependent and -independent pathways.

Amino Acids↗
PubMed1997

In situ measurement of glutamate concentrations in the periportal, intermediate, and pericentral zones of rat liver.

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (> 96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 +/- 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (approximately 14 mM) and intermediate and pericentral zones approximately 13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to approximately 15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of approximately 5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to approximately 8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.

Age Factors↗
PubMed1996

The role of the intralysosomal pH in the control of autophagic proteolytic flux in rat hepatocytes.

Current methods to estimate changes in intralysosomal pH in hepatocytes do not discriminate between lysosomes and other intracellular acidic compartments. To obtain selective information on the change in lysosomal function in response to a change in lysosomal pH we have used the liberation of fluorescent 4-methoxy-2-naphthylamide from low concentrations of lysyl-alanyl-4-methoxy-2-naphthylamide, a substrate of lysosomal dipeptidylpeptidase II. Using permeabilized and intact hepatocytes, the activity of this enzyme in response to manipulations meant to increase the intralysosomal pH was compared with intralysosomal protein degradation and with the accumulation of [14C]chloroquine as a pH indicator of intracellular acidic compartments. The data show that changes in intralysosomal pH are indicated by changes in dipeptidylpeptidase II activity and that these are mainly due to a pH-dependent change in substrate accumulation in the lysosomes. Subsequently, the method was applied to establishing the extent to which an increase in intralysosomal pH can contribute to the inhibition of autophagic proteolysis in intact hepatocytes caused by a decrease in intracellular ATP, by an increase in amino acid concentration and by hypo-osmotic cell swelling. The following observations were made. (a) Moderate changes in intracellular ATP do not affect the lysosomal pH. (b) Hypo-osmotic cell swelling, which promotes inhibition of proteolysis by amino acids in freshly isolated hepatocytes, does not affect the lysosomal pH. (c) In addition to their known inhibitory effect on autophagic sequestration, amino acids (leucine in particular) can increase the lysosomal pH and thus inhibit intralysosomal protein degradation directly. (d) Low concentrations of the acidotropic agent methylamine increase the lysosomal pH without having an effect on autophagic proteolytic flux. It is concluded that autophagic proteolysis is not controlled by changes in the lysosomal pH.

2-Naphthylamine↗
PubMed1996

Response of hepatic amino acid consumption to chronic metabolic acidosis.

In a previous paper, we showed that an inhibition of amino acid transport across the liver plasma membrane is responsible for the decrease in urea synthesis in acute metabolic acidosis. We have now studied the mechanism responsible for the decline in urea synthesis in chronic acidosis. Chronic metabolic acidosis and alkalosis were induced by feeding three groups of rats HCl, NH4Cl, and NaHCO3 (8 mmol/day) for 7 days. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences were calculated. The rates of urinary urea and NH4+ excretion were also determined. Hepatic amino acid consumption was lower in both HCl and NH4Cl acidosis compared with NaHCO3-fed rats. Glutamine release was not different in the three conditions. Because intrahepatic concentrations of amino acids and intracellular protein degradation were similar under these conditions, it can be concluded that at low blood pH amino acid catabolism may be inhibited and might explain the observed decrease in urea excretion in HCl, but not NH4Cl, acidosis; urea excretion was comparable in the NH4Cl and NaHCO3 groups presumably because the increased NH4+ load in the former group was processed, uninhibited, to urea. Amino acids not used by the liver in acidosis could account for the 25-fold increase in NH4+ excretion in HCl and NH4Cl compared with alkalosis (P < 0.05). These findings indicate that urea synthesis is decreased in chronic HCl acidosis. They show that urea synthesis is controlled in chronic, as in acute, acidosis by amino acid uptake by the liver and/or intrahepatic degradation and that the ornithine cycle per se has only minor control of acid-base homeostasis.

Acidosis↗
PubMed1995

Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes.

In rat hepatocytes, autophagy is known to be inhibited by amino acids. Insulin and cell swelling promote inhibition by amino acids. Each of the conditions leading to inhibition of autophagic proteolysis was found to be associated with phosphorylation of a 31-kDa protein that we identified as ribosomal protein S6. A combination of leucine, tyrosine, and phenylalanine, which efficiently inhibits autophagic proteolysis, was particularly effective in stimulating S6 phosphorylation. The relationship between the percentage inhibition of proteolysis and the degree of S6 phosphorylation was linear. Thus, inhibition of autophagy and phosphorylation of S6 are under the control of the same signal transduction pathway. Stimulation of S6 phosphorylation by the presence of amino acids was due to activation of S6 kinase and not to inhibition of S6 phosphatase. The inhibition by amino acids of both autophagic proteolysis and autophagic sequestration of electro-injected cytosolic [14C]sucrose was partially prevented by rapamycin, a compound known to inhibit activation of p70 S6 kinase. In addition, rapamycin partially inhibited the rate of protein synthesis. We conclude that the fluxes through the autophagic and protein synthetic pathways are regulated in an opposite manner by the degree to which S6 is phosphorylated. Possible mechanisms by which S6 phosphorylation can cause inhibition of autophagy are discussed.

Adenine↗
PubMed1995

Changes in hepatic nitrogen balance in plasma concentrations of amino acids and hormones and in cell volume after overnight fasting in perinatal and adult rat.

Important regulatory factors of intrahepatic protein synthesis and proteolysis are amino acids, glucagon, insulin, and cell volume. We have investigated the changes in these factors with development and after an overnight fast and evaluated their contribution to changes in the hepatic nitrogen balance in vivo. In the fed state, glucagon levels were highest in suckling animals and gradually declined in older rats, whereas the concentration of insulin increased during development. The amino acid concentrations in liver and plasma declined during the suckling period to levels that in vitro are highly permissive for induction of autophagic proteolysis. In all age groups investigated, fasting was associated with a drop in hepatic protein content, together with a marked decrease in hepatocellular volume and insulin concentrations. On the other hand, glucagon concentrations and the concentration of many amino acids in plasma and liver responded to fasting with a pronounced decrease in perinatal and suckling animals, but this response had become blunted at weaning and had disappeared in adult animals. These findings suggest that insulin and/or hepatocellular volume are more likely candidates as short-term physiologic regulators of the hepatic nitrogen balance than are glucagon or amino acids. In glucose-supplemented fetuses, high levels of insulin could not compensate for a decreased hepatocellular volume in averting a catabolic state, suggesting that cell volume is the more important factor. Although our study cannot discriminate between the effects of fasting on protein synthesis and degradation, our findings show unequivocally that, for a rapid growth of the liver, suckling animals have to be fed around-the-clock.

Amino Acids↗
PubMed1995

In vivo amino acid fluxes in regenerating liver after two-thirds hepatectomy in the rat.

BACKGROUND/AIMS: Recent reports in the literature suggest that liver cell swelling following amino acid influx exerts anabolic and anti-catabolic effects. We have tested the possibility that rapid liver growth after partial hepatectomy is promoted by an increased amino acid-influx and that this is associated with an increased hepatic water content and a decreased rate of proteolysis. METHODS: Two-thirds hepatectomy was performed in rats. Plasma liver flow and amino acid fluxes were measured after 24 or 48 h. RESULTS: Plasma liver flow was increased 24 and 48 h after partial hepatectomy or sham-operation in pair-fed animals. At these time points, in both groups there was a specific two- to threefold increased net hepatic uptake of the amino acids alanine and glycine, both being transported by the sodium-coupled amino acid transport system A/ASC. No changes in uptake of system N transported amino acids were observed. Both in partially hepatectomized and sham-operated pair-fed animals, the hepatic uptake of alanine and glycine was accompanied by a minor increase in tissue water (from 68 to 70%). Proteolysis, measured by leucine efflux, was only reduced in regenerating livers. CONCLUSIONS: We conclude that cell swelling is not an important factor in the stimulation of net protein synthesis during liver regeneration.

Amino Acids↗
PubMed1994

Carbamoyl phosphate and ureagenesis are not involved in amino-acid-stimulated glycogenesis.

Amino acids are known to stimulate glycogen synthesis via an increase in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959]. It has recently been postulated, however, that carbamoyl phosphate, an intermediate of ureagenesis, can function as a substrate for glucose phosphorylation via carbamoyl-phosphate:glucose phosphotransferase activity of the glucose-6-phosphatase system. This hypothesis would account for the stimulation of glycogenesis by amino acids such as glutamine and proline [Bode, A. M. & Nordlie, R. C. (1993) J. Biol. Chem. 268, 16298-16301]. To further examine the role carbamoyl phosphate may play in glycogenesis, isolated hepatocytes were incubated under a variety of conditions to manipulate ureagenesis, glycogenesis and carbamoyl-phosphate levels. Our data indicate that carbamoyl-phosphate levels do not correlate with amino-acid-stimulated glycogenesis and that ureagenesis and glycogenesis are not competing metabolic pathways.

Amino Acids↗
PubMed1994

Acute acidosis inhibits liver amino acid transport: no primary role for the urea cycle in acid-base balance.

To examine further the role of the liver in acid-base homeostasis, we studied hepatic amino acid uptake and urea synthesis in rats in vivo during acute acidosis and alkalosis, induced by infusion of 1.8 mmol of HCl or NaHCO3 over 3 h. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences of amino acids and urinary urea and NH4+ excretion were measured. In acidosis, urinary urea excretion was reduced 36% (P < 0.01), whereas urinary NH4+ excretion increased ninefold (P < 0.01), but the sum of urea and NH4+ excretion was unchanged. Total hepatic amino acid uptake, as determined from arteriovenous differences, was decreased by 63% (P < 0.01) in acidosis, with the major effect being noted with alanine and glycine. Only glutamine was released in both acidosis and alkalosis but was not significantly different in the two conditions. Since intracellular concentrations of readily transportable amino acids were not different at low pH despite accelerated protein degradation, these results indicate that hepatic amino acid transport was inhibited markedly and sufficiently to explain the observed decrease in urea synthesis. Total hepatic vein amino acid content was greater in acidosis than alkalosis (P < 0.01). Directly or indirectly, by conversion to glutamine elsewhere, these increased amino acids were degraded in kidney and accounted for the ninefold increase in urinary NH4+ excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1993

Cell swelling and the sensitivity of autophagic proteolysis to inhibition by amino acids in isolated rat hepatocytes.

In the isolated perfused rat liver, autophagic proteolysis is inhibited by hypo-osmotic perfusion media [Häussinger, D., Hallbrucker, C., vom Dahl, S., Lang, F. & Gerok, W. (1990) Biochem. J. 272, 239-242]. Here we report that in isolated hepatocytes, incubated in the absence of amino acids to ensure maximal proteolytic flux, proteolysis was not inhibited by hypo-osmolarity while the synthesis of glycogen from glucose, a process known to be very sensitive to changes in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959], was stimulated under identical conditions. However, in isolated hepatocytes, hypo-osmolarity increased the sensitivity of autophagic proteolysis to inhibition by low concentrations of amino acids. The anti-proteolytic effect of hypo-osmolarity in our experiments was not due to stimulation of amino-acid transport into the hepatocytes: neither the consumption of most amino acids, nor the rate of urea synthesis was appreciably affected by hypo-osmotic incubation conditions. In the course of these studies we also found that hypo-osmolarity increased the affinity of protein synthesis for amino acids. In the presence of amino acids the intracellular level of ATP was not much affected. However, because of cell swelling under these conditions the intracellular concentration of ATP decreased. It is proposed that a small part of the inhibition of proteolysis by amino acids may be due to this fall in ATP concentration.

Adenosine Triphosphate↗
PubMed1993

Effects of intracellular amino acid concentrations, cyclic AMP, and dexamethasone on lysosomal proteolysis in primary cultures of perinatal rat hepatocytes.

We have studied factors regulating the rate of protein degradation in cultured hepatocytes obtained from 17-day-old fetal, 7-day-old suckling, and 20-day-old weanling rats. At all three stages of development 60-70% of protein degradation was sensitive to inhibition by amino acids and 3-methyladenine, an inhibitor of macroautophagy, indicating a major role of the lysosomes in proteolysis under these conditions. A combination of dibutyryl cyclic AMP and dexamethasone strongly stimulated proteolysis in hepatocytes from weanling, but not from fetal and suckling rats. The stimulatory effect of these compounds was eliminated at high amino acid concentrations in the culture medium. Cultured perinatal hepatocytes responded to exposure to dibutyryl cyclic AMP and dexamethasone by de novo synthesis of mRNA for carbamoyl-phosphate synthase and for phosphoenolpyruvate carboxykinase, demonstrating that the developmental change in the effect of dibutyryl cyclic AMP and dexamethasone on proteolysis was due to a developmental change in the regulation of proteolysis. An analysis of the changes in intracellular amino acid concentrations in response to variations in the extracellular amino acid concentrations at all three stages of development showed that of all amino acids that could be identified, only Ile, Leu, Lys, Phe, and Tyr are implicated as possible regulators of hepatic proteolysis. Dibutyryl cyclic AMP and dexamethasone did not affect the intracellular concentrations of these amino acids, showing that hormonal regulation of proteolysis is not mediated by changes in intracellular concentrations of these amino acids. It is concluded that the lack of sensitivity of the proteolytic system to catabolic hormones in the period around birth, combined with higher circulating plasma amino acid concentrations, are mechanisms contributing to the low rate of intrahepatic proteolysis in vivo in the perinatal period and thus to the rapid growth of the liver in this period.

Adenine↗
PubMed1992

Autophagic degradation of peroxisomes in isolated rat hepatocytes.

Degradation of the peroxisomal enzymes fatty acyl-CoA oxidase and catalase was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. Hepatocytes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximide to inhibit protein synthesis. (1) Degradation of the two peroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes. This increased degradation of the peroxisomal enzymes was almost completely prevented by 3-methyladenine, an inhibitor of macroautophagic sequestration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (C16:0) and a very-long-chain (C26:0) fatty acid, but not by C12:0, a medium-chain fatty acid, or by C8:0, a short-chain fatty acid. These results provide direct evidence for the proposal that autophagic sequestration can be highly selective [(1987) Exp. Mol. Pathol. 46, 114-122]. It is concluded that preferential autophagy of peroxisomes is prevented when these organelles are supplied with their fatty acid substrates.

Animals↗