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.

In the isolated perfused rat liver, autophagic proteolysis is inhibited by hypo-osmotic perfusion media [Haussinger, D., Hallbrucker, C . , vom Dahl, S., Lang, F. & Gerok, W. (1990) Biochem. J. 272, 239-2421. 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. B i d . Chein. 265, 955 -9591, 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.Evidence is rapidly accumulating that an increase in the volume of hepatocytes, in response either to intracellular amino acid accumulation or to a decrease in the osmolarity of the extracellular fluid, has anabolic and anti-catabolic effects. Thus, an increase in cell volume results in increased glycogen synthesis [l, 21, increased lipogenesis [3, 41, increased polyamine synthesis [5], increased metabolism of some amino acids [6, 71, and in decreased glycogenolysis [8].Haussinger and coworkers [9-111 demonstrated that in the isolated perfused rat liver an increase in cell volume also inhibited protein breakdown and that this phenomenon could account for, at least in part, the well known property of some amino acids to inhibit this process. However, Car0 [12] showed that in isolated rat hepatocytes, an increase in cell volume did not inhibit proteolysis, or even slightly stimulated it, when flux through the autophagic pathway was maximal, i.e. in the absence of added amino acids. Intrigued by this puzzling difference in results, which was not discussed [ll], we decided to investigate this property of isolated hepatocytes in more detail. We have now found in rat hepatocytes that, although a decrease in medium osmolarity per se does not appreciably affect proteolysis, it does increase the sensitivity of proteolysis to inhibition by amino acids. MATERIALS AND METHODSRat hepatocytes were isolated from 20-24-h fasted male Wistar rats (200-250 g) as in [13].Hepatocytes (5 mg dry cells/ml) were incubated in closed 25-ml p...

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)

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.

ARSTRImportant 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 glucosesupplemented 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. (Pediatr Res 38: 1018-1025, 1995)The cellular protein content is regulated by modulation of the rates of synthesis and/or degradation. The nutritional and hormonal status have been shown to be of paramount importance as modulators of the rate of synthesis and degradation of proteins in the liver (1-7). In vivo studies in which the effect of refeeding after prolonged periods of starvation (2-5 d) was studied (8-10) and in vitro studies with perfused livers (cf. Ref. I), freshly isolated (6,7,11,12) or cultured hepatocytes (13-15) have identified amino acids and the hormones insulin and glucagon as major determinants of hepatic lysosomal proteolysis and protein synthesis. High ambient concentrations of amino acids and insulin suppress intrahepatic proteolysis (1, 6, 7) and stimulate protein synthesis (16-19), whereas glucagon exerts the opposite effect (1,13,14,20). More recently, it was proposed that lysosomal proteolysis in rat liver is also regulated by changes in the volume of the hepatocyte, an increase in cell volume being inhibitory (21, 22). As indicated, most of these findings were obtained from in vitro studies, involving strong manipulation of the nutritional and hormonal environment of the hepatocytes. In this respect, we had previously observed in cultured hepatocytes that intrahepatic lysoso...