Inhibition of autophagy by benzyl alcohol

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Biochimica et Biophysica A cta, 800 (1984) 140 144

Elsevier BBA 21799

I N H I B I T I O N O F A U T O P H A G Y BY B E N Z Y L A L C O H O L BJORN GRINDE University of Oslo, Department of Biology, Division of Zoology, P.O. Box 1050, Blindern, N-0316 Osh) 3 (Norway)

(Received February 6th, 1984)

Key words: Autophagy," Protein degradation; Benzyl alcohol inhibition; (Rat Hepatocyte)

Benzyi alcohol caused a rather complete and selective inhibition of the methylamine sensitive (i.e., the putative lysosomal) pathway of protein degradation in isolated rat hepatocytes. The effect was found to be entirely reversible within 30 min of removing the agent. A morphometric examination of electron micrographs revealed that the inhibition of lysosomal protein degradation coincided with a block in the formation of autophagic vacuoles. The number of acidic vacuoles (i.e., vacuoles induced to swell by adding methylamine) was not drastically reduced.

Introduction Benzyl alcohol is a well-known membrane-perturbing agent. Experiments with various membrane model systems show that it decreases the lipid-phase transition temperature even at concentrations below 2 m M [1], at 7.5 m M it increases the membrane thickness [2], and at concentrations above 10 m M it increases membrane fluidity, as indicated by various methods [1,3-5]. At a concentration level of 10 mM, benzyl alcohol inhibits the uptake and the degradation of asialo-glycoprotein in isolated rat hepatocytes, as demonstrated by Tolleshaug and Berg [6]. They conclude that the effect on degradation is due to inhibition of fusion between endosomes and lysosomes, while the effect on uptake follows a reduced rate of receptor recycling, which again is a consequence of the inhibitory effect on fusion. Weak bases, such as methylamine, accumulate in acidic intracellular vacuoles (e.g., lysosomes) thereby disrupting the activity of these vacuoles. Methylamine is believed to cause a fairly complete and specific inhibition of the lysosomal degradation of endogenous protein in rat hepatocytes cultured as described by Seglen and colleagues [7-10] 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

and in rat embryo fibroblasts as decribed by Amenta and Brocher [11]. The basis for this assumption is that other agents that influence lysosomal proteolysis by totally different mechanisms (e.g., proteinase inhibitors, amino acids, microtubulus poisons, etc.) have little or no effect on the methylamine-insensitive degradation of proteins. The effect of methylamine is believed to be a consequence of either a dramatic swelling of acidic vacuoles [7,12], or an increment in their p H [13,14]. The swelling apparently gives rise to a general decline in cellular membrane transport [15,16]. I have employed methylamine as a diagnostic tool to indicate whether the effect of benzyl alcohol is on lysosomal or non-lysosomal proteolysis. The present data indicate that benzyl alcohol, at a concentration of 10 mM, causes a block in the formation of autophagic vacuoles, and may therefore be a useful tool in the study of this process.

Methods Cells

Hepatocytes were prepared from the livers of 16-h-fasted male Wistar rats (220-260 g), using

141 the collagenase perfusion m e t h o d [17]. The cells were incubated in suspension as previously described [8], the suspension buffer [17| employed being modified by the addition of 15 m M pyruvate and 10 m M glucose. Viability was routinely tested. b o t h before and after incubation, and found to be in the range 90-95%, according to the T r y p a n blue exclusion test. Biochemicals were purchased from Sigma.

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Electron microscopy The cells were fixed with 2% glutaraldehyde (for 4 h at r o o m temperature) and post-fixed (for 1 h at 4 ° C ) with 1% OsO 4. Ultrathin sections were stained with uranyl acetate and lead citrate. For a more detailed description of the preparation of electron micrographs see Ref. 18. M o r p h o m e t r y was carried out on micrographs at 7800 × magnification using a superimposed lattice with 0.55 ~tm intersection points as described by Reith et al. [19]. The numerical density of vacuoles is related to the area of cytoplasm on the micrographs, the volume fraction of vacuoles is related to the volume of cytoplasm.

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Protein degradation The degradation of endogenous protein was measured as the release of [14C]valine (No. 10139 from ICN, CA) from protein prelabelled for 24 h in vivo. The procedure is described in more detail in Refs. 7 and 8. Unlabelled valine (5 raM) was added to prevent reutilization of [14C]valine.

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Fig. 1. Effect of benzyl alcohol on protein degradation in the absence or presence of methylamine. Hepatocytes were prelabelled for 24 h in vivo with [14C]valine. The cells were incubated for 2 h with various concentrations of benzyi alcohol, in the absence (©) or presence (O) of methylamine (12 mM). The net release of [14C]valineduring the incubation period was measured, and expressed as percentage of the initial protein radioactivity. Each point is the mean of two cell samples, the two values are depicted (as crosses) where they fall outside the symbols. a quite short (approx. 10 min) lag period before maximal effect was achieved. The effect of benzyl alcohol was found to be completely reversible. Some 30 rain after the agent had been removed from the medium, the rate of degradation was the i

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Results

Biochemical analysis Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from cells prelabelled for 24 h in vivo. In Fig. 1 the effect on protein degradation of various concentrations of benzyl alcohol has been tested, both in the absence and presence of the lysosomotropic weak base methylamine. As can be seen, at 10 m M benzyl alcohol blocked methylamine-sensitive proteolysis almost completely, while having only a slight effect on methylamine-resistant degradation. Time curves for the effect of benzyl alcohol on protein degradation (Fig. 2), using cells preincubated with or without benzyl alcohol, indicated

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Fig. 2. Time-course and reversibility of the benzyl alcohol effect, Hepatocytes were prelabelled for 24 h in vivo with [14C]valine. The isolated cells were preincubated for 20 min with (zx,A) or without (O, e) benzyl alcohol (10 mM), washed twice and then reincubated for up to 2 h in the absence (O, zx) or presence (O, -) of benzyl alcohol (10 mM). The net release of [14C]valine during the final incubation was measured, and expressed as percentage of the initial protein radioactivity. Each point is the mean of two cell samples, the two values are depicted (as crosses) where they fall outside the symbols.

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same as (or slightly higher than) that in cells not exposed to benzyl alcohol.

Morphometric analysis A morphometric analysis of vacuoles belonging to the autophagic/lysosomal system was undertaken to obtain information on the mechanism of the benzyl alcohol effect. I distinguished between two types of autophagic/lysosomal vacuole. Type 1, here referred to as autophagosomes, has a content that appears relatively unaffected by hydrolytic enzymes, i.e., it consists of recognizable, cytoplasmatic components. Type 2, here referred to as secondary lysosomes (a term meant to include autolysosomes), has a content that is clearly changed compared to the surrounding cytoplasm. In the presence of methylamine, many large (swollen) vacuoles with very little visible content emerged. These vacuoles were obviously acidic, otherwise methylamine would not have caused them to swell. Their lack of recognizable content, however, made it difficult to deduce their origin. In my analysis they have nevertheless been classified as secondary lysosomes, but, as will be discussed below, they may not all be lysosomes. Dense bodies are sparse in preparations of hepatocytes [9], and as no appreciable variation was seen between the different additions, this type of lysosomal vacuole was not included in the analysis. Examples of type 1 and 2 vacuoles are shown in Fig. 3; for more detailed pictures of these vacuoles consult Refs. 9 and 18. Amino acids are well-known inhibitors of autophagy [9,20]. As can be seen from Table I, benzyl alcohol caused an even more drastic reduction than amino acids in the number and volume of both autophagosomes and secondary lysosomes. In the presence of benzyl alcohol the number and volume of vacuoles were approximately the same before and after incubating the cells. Compared with cells incubated without any addition, the number of vacuoles in benzyl alcohol-treated cells was only a fraction. Incubation with methylamine for 1 h caused a drastic increase in the quantity of vacuoles, particularly in the volume of secondary lysosomes. Pretreating the cells for 30 min with benzyl alcohol prevented the increase in autophagosomes due to

Fig. 3. Classification of autophagic vacuoles. Hepatocytes were incubated for 90 min at 37°C, then prepared for electron microscopy. Autophagosomes are labelled 1, secondary lysosomes 2 (see text for a description of this classification). The cell was incubated in the absence of methylamine (A) or in the presence of 12 mM methylamine for the last 60 rain (B). Pictures of cells treated with benzyl alcohol are not shown, as the agent caused no change in the appearance of the cells, except for the decrease in autophagic vacuoles. Magnification is 7020 ×.

methylamine. Benzyl alcohol, however, only caused a 25% lessening of the enormous increase in secondary lysosomes seen in cells incubated with methylamine. The immediate appearance of methylaminetreated cells was very similar in the presence or absence of benzyl alcohol. In both cases, large vacuoles, relatively void of visible content, dominated their appearance. The vacuoles were possibly even more empty in cells incubated with benzyl alcohol; this difference, however, if existent, was not conspicuous.

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TABLE I MORPHOMETRIC ANALYSIS OF T H E M O R P H O L O G I C EFFECT OF BENZYL ALCOHOL Freshly isolated hepatocytes were prepared for electron microscopy either before incubation or after 90 min of incubation. Additions were made at the following concentrations: benzyl alcohol, 10 mM; methylamine, 12 mM; and a complete amino acid mixture as described in Ref. 9. Methylamine was added after 30 min of incubation, the other substances before incubating the cells. Morphometry was performed on electron micrographs at 7800 × magnification. Each value represents the mean 4- S.E. of 20 different ceils. Levels of significance of differences are according to Student's t test, the noted levels stand for comparison between both numerical density and volume fractions. Numerical density 1 Autophagosomes A Before incubation B Incubated control C Benzyl alcohol D Methylamine E Benzyl alcohol + methylamine F Amino acids 2 Secondary lysosomes A Before incubation B Incubated control C Benzyl alcohol D Methylamine E Benzyl alcohol + methylamine F Amino acids

0.4+ 3.0 + 0.4 + 5.5 +

Volume fraction (mm3/cm3)

Comparison

Significance level ( a )

0.1 0.4 0.2 0.6

0.5 + 0.1 8.0 + 1.2 0.6 ___0.3 16.7 + 2.4

C vs. B D vs. B

0.0005 0.001

1.0 + 0.2 0.5 +0.2

1.5 + 0.3 0.8+0.5

E vs. D F vs. B

0.0005 0.0005

1.0 + 0.3 5,6 + 0.8 0.8 4- 0.3 24.1 4-1.5

1.4 + 0.5 23.3 _4-_4.8 1.9 4- 0.7 135.1 4- 9.4

C vs. B D vs. B

0.0005 0.0005

19.8 _ 1.8 2.1 4- 0.6

106.7 + 10.6 4.6 5:1.3

E vs. D F vs. B

0.05 0.0005

Discussion

In the present paper I have described results indicating that benzyl alcohol causes a seemingly complete block of autophagic sequestration, implying that this agent may be a convenient tool for investigating autophagy. Benzyl alcohol has previously been indicated to inhibit the fusion of endosomes with lysosomes [6]. From the present data it is not possible to decide whether benzyl alcohol would also influence the fusion of autophagosomes with lysosomes, as the agent appeared to block the autophagic process at the earlier, sequestrating step. Amino acids cause an approx. 80% inhibition of methylamine-sensitive proteolysis, with a negligible effect on methylamine-insensitive degradation [9]. Benzyl alcohol was an even more powerful inhibitor, selectively blocking almost all methylamine-sensitive degradation (Fig. 1). These biochemical data are reflected in Table I, showing

that benzyl alcohol decreased the level of autophagic vacuoles even more than did amino acids. The effect of benzyl alcohol adds good evidence to the notion that methylamine causes a rather complete and selective inhibition of lysosomal degradation of endogenous protein. Methylamine inhibits the spreading of hepatocytes over the surface of culture dishes [15]. Apparently a flow of membrane between the plasma membrane and the intracellular membrane system is necessary for the spreading of the cells on a substratum, and the swelling of acidic vacuoles interferes with this flow [16]. If benzyl alcohol retards the fusion of intracellular vesicles, it might therefore be expected to delay the spreading of the cells. I found (unpublished data), employing a method similar to that of Seglen and Gordon [15], that benzyl alcohol was an even more potent inhibitor of cell spreading than methylamine. The absolute increase in the apparent number

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of secondary lysosomes, caused by methylamine, was the same with cells preincubated and incubated in the presence or absence of benzyl alcohol (Table I). This indicates that benzyl alcohol treatment did not result in any drastic reduction in the number of acidic vacuoles, implying some selectivity in its membrane-perturbing effect. Methylamine may cause a real build-up in the number of acidic vacuoles, for example, due to an inhibition of their processing. Very likely, however, most of the swollen vacuoles are also present in the absence of methylamine, but not recognized as part of the lysosomal system due to their small size. It should be pointed out that the cells most likely contain acidic vacuoles other than lysosomes. Endosomes have recently been shown to acquire an ATP-driven proton pump, and become acidic, before they acquire lysosomal enzymes [21-23]. The acidic vacuoles seen in cells treated with both benzyl alcohol and methylamine may be assumed to be either primary lysosomes or to belong to the endocytotic system. Most of the methylamine-swollen vacuoles contained little recognizable material, whether the cells were incubated in the presence or absence of benzyl alcohol. Their classification was therefore difficult. The autophagic/lysosomal degradation of protein depends on energy [7]. The effect of benzyl alcohol might therefore be a result of a reduction of the ATP level. It has been demonstrated, however, that a 1 h incubation of isolated rat hepatocytes with 10 m M benzyl alcohol results in a mere 10% drop in A T P concentration [6]. To summarize, the membrane-perturbing agent benzyl alcohol (at 10 mM) seems to have two distinct effects on isolated rat hepatocytes. It blocks the sequestration step in autophagy, and it inhibits the fusion of lysosomes with other intracellular vacuoles. The agent may be relatively selective for these two processes.

Acknowledgements This work was supported by a grant from A / S Norsk Varekrigsforsikrings Fond. I would like to

thank Solve Stenmark for making electron micrographs of the cells.

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