3-Methyladenine is generally used as an inhibitor of autophagy [P. 0. Seglen & P. B. Gordon (1982) Proc.Natl Acad. Sci. U S A 79, . Using isolated hepatocytes, we observed that 3-methyladenine has other effects as well.1. 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by CAMP, which slightly increased in the presence of 3-methyladenine.2. Addition of 3-methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3-methyladenine in isolated lysosomes.3-Methyladenine is a powerful inhibitor of autophagy and primarily interferes with the formation of autophagosomes [1 -31. In the course of a study of factors involved in the control of proteolysis in isolated rat hepatocytes we observed that 3-methyladenine has other effects on cellular functions in addition to its action as an inhibitor of autophagy. Since 3-methyladenine is now widely used as a specific inhibitor of autophagy, the interpretation of results obtained with this compound should take such additional effects into account. MATERIALS AND METHODS HepatocytesHepatocytes were isolated from the livers of male Wistar rats (200-250 g) using the method of Berry and Friend [4] with the modifications described by Groen et al. [5].Hepatocytes (5 -10 mg dry mass/ml) were incubated at 37 "C in 25-ml plastic counting vials with Krebs-Ringer bicarbonate buffer, containing 1.3 mM Ca2+ and the additions mentioned in the legends to the figures and tables. The gas phase was 95% O2 plus 5% C 0 2 and the final volume was 2 -4 ml. Incubations were terminated by the addition of cold sulfosalicylic acid (final concentration 4%, mass/vol.) for the analysis of valine, or by the addition of HC104 (final concentration 3.5%, mass/vol.) for measurement of glucose and lactate. Samples for valine analysis were brought to pH 2.2 with 3 M LiOH. Perchloric acid extracts were neutralised to pH 7 with a mixture of 2 M KOH and 0.3 M Mops.For determination of the intracellular content of metabolites, the cells were centrifuged through a layer of silicone oil (Wacker, AR 200: AR 20 = 3: 2) into a layer of 14% HC104.Proteolysis was measured as the rate of appearance of valine in the incubation medium [6].

It has been shown previously that the inhibition of autophagic proteolysis in liver by a physiological mixture of amino acids can be mimicked completely by addition of leucine in combination with alanine [Leverve, X. M., Caro, L. H. P., Plomp, P. J. A. M. and Meijer, A. J. (1987) FEBS Lett. 219,. We have now further defined conditions which lead to this inhibition.Isolated rat hepatocytes were incubated in the perifusion system in which the cells can be maintained at a steady state in the presence of low amino acid concentrations.Combinations of leucine (0.5 mM) with either alanine, glutamine, asparagine or proline (2 mM) inhibited proteolysis by 40-50%. Under these conditions, both in the absence and presence of the transaminase inhibitor, aminooxyacetate, a correlation was found between the extent of inhibition of proteolysis and the sum of the total intracellular amounts of aspartate and glutamate.Inhibition of proteolysis by leucine and leucine analogues did not correlate with their ability to activate glutamate dehydrogenase.Intracellular protein degradation is strongly stimulated during food deprivation. In this way amino acids are generated which can be used for synthesis of essential proteins and other nitrogen-containing compounds, for gluconeogenesis and ketogenesis and as oxidisable substrates for ATP production. In liver, starvation-induced proteolysis, which occurs via autophagy, accounts for at least 70% of total proteolysis Hepatic autophagic protein breakdown can be regulated by hormones: there is inhibition by insulin and stimulation by glucagon [4]. Experiments carried out with the isolated perfused liver [5] and with isolated hepatocytes [6] have shown that flux through the autophagic pathway is maximal in the absence of amino acids and is strongly decreased by high concentrations of amino acids. However, the mechanism by which amino acids inhibit autophagy is still obscure (see [7] for review). According to Mortimore and coworkers (e. g. [4,8, 91) a regulatory group of amino acids containing leucine, tyrosine, glutamine, proline, histidine, tryptophan and methionine, acts together with alanine (which does not inhibit on its own) as a synergistic coregulator in inducing the antiproteolytic response. Using perifused hepatocytes, we found previously that a combination of low concentrations of alanine and leucine alone strongly and synergistically inhibits autophagic proteolysis [lo]. Since leucine is not degraded in rat liver [Il, 121, inhibition of proteolysis by leucine must be due to a direct effect of this amino acid. Inhibition by alanine, however, is prevented by the transaminase inhibitor aminooxyacetate, indicating that a catabolite of alanine (which could be glutamate or aspartate) is required for this effect [lo].[I -31. We have now studied the effect of other amino acids, which in the course of their metabolism give rise to rapid production of intracellular glutamate and aspartate, in order to test the hypothesis that, in addition to leucine, intracellular glutamate and/or aspartate ...

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