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].

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made.1. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite.2. There was a linear relationship between the autophagic sequestration of cytosolic ['4C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations.3. ATP depletion did not cause release of ['4C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels.4. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content.5. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.Autophagic degradation of endogenous liver cell protein is an energy-dependent process. This conclusion can be drawn on the basis of experiments with rat liver slices or with isolated rat hepatocytes, showing that proteolysis via autophagy is greatly diminished under conditions where mitochondria1 ATP production is strongly, if not completely, suppressed by high concentrations of inhibitors [l -31 or by replacing oxygen with nitrogen [l, 4, 51. Although these experiments do, indeed, suggest that ATP is required for proteolysis, they do not give any information about the steps in the autophagic pathway that require ATP; nor do they give information on the sensitivity of these steps towards small, more physiological changes in intracellular ATP concentration.The experiments described in this paper were designed to answer these questions. As an experimental system we used isolated rat hepatocytes, incubated under amino-acid-free conditions to ensure maximal rates of autophagic protein degradation [3, 61. MATERIALS AND METHODS Isolation of hepatocytesHepatocytes were isolated from 20-h-starved male Wistar rats (200-280 g) either according to Berry and Friend [7] with the modifications described by Groen et al. [8] or, in the experiments in which autophagic sequestration of intracellular sucrose was measured, by the method of Seglen [9]. Gordon and Seglen [lo, 111: hepatocytes were electropermeabilized with five high-voltage pulses and incubated with [14C]sucrose (5-10 pCi/ml) for 1 h at 0"C, followed by a resealing period of 30 min at 37°C and washing at 0 "C to remove extracellular radioactivity.Autophagic sequestration of the intracellular ['4C]sucrose was measured exactly as described by Gordon et al. [1...

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The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and l-2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.

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