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...

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.

No abstract

The oxidation of ethanol by isolated liver cells from starved rats is limited by the rate of removal of reducing equivalents generated in the cytosol by alcohol dehydrogenase. Evidence is presented suggesting that, in these cells, transfer of reducing equivalents from the cytosol to the mitochondria is regulated by the intracellular concentrations of the intermediates of the malate-aspartate and glycerol 3-phosphate cycles, as well as by flux through the respiratory chain. In liver cells isolated from fed rats, the availability of substrate increased the cell content of intermediates of the hydrogen-transfer cycles, and enhanced ethanol uptake. Under these conditions, ethanol consumption is limited by the availability of ADP for oxidative phosphorylation.