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Originally published In Press as doi:10.1074/jbc.M502456200 on May 9, 2005

J. Biol. Chem., Vol. 280, Issue 27, 25864-25870, July 8, 2005
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Ketone Bodies Stimulate Chaperone-mediated Autophagy*

Patrick F. Finn{ddagger} and J. Fred Dice

From the Department of Molecular and Cellular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Received for publication, March 4, 2005 , and in revised form, April 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chaperone-mediated autophagy (CMA) is a selective lysosomal protein degradative process that is activated in higher organisms under conditions of prolonged starvation and in cell culture by the removal of serum. Ketone bodies are comprised of three compounds ({beta}-hydroxybutyrate, acetoacetate, and acetone) that circulate during starvation, especially during prolonged starvation. Here we have investigated the hypothesis that ketone bodies induce CMA. We found that physiological concentrations of {beta}-hydroxybutyrate (BOH) induced proteolysis in cells maintained in media with serum and without serum; however, acetoacetate only induced proteolysis in cells maintained in media with serum. Lysosomes isolated from BOH-treated cells displayed an increased ability to degrade both glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, substrates for CMA. Isolated lysosomes from cells maintained in media without serum also demonstrated an increased ability to degrade glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A when the reaction was supplemented with BOH. Such treatment did not affect the levels of lysosome-associated membrane protein 2a or lysosomal heat shock cognate protein of 70 kDa, two rate-limiting proteins in CMA. However, pretreatment of glyceraldehyde-3-phosphate and ribonuclease A with BOH increased their rate of degradation by isolated lysosomes. Lysosomes pretreated with BOH showed no increase in proteolysis, suggesting that BOH acts on the substrates to increase their rates of proteolysis. Using OxyBlotTM analysis to detect carbonyl formation on proteins, one common marker of protein oxidation, we showed that treatment of substrates with BOH increased their oxidation. Neither glycerol, another compound that increases in circulation during prolonged starvation, nor butanol or butanone, compounds closely related to BOH, had an effect on CMA. The induction of CMA by ketone bodies may provide an important physiological mechanism for the activation of CMA during prolonged starvation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For eukaryotic cells to maintain homeostasis, a balance between protein synthesis and proteolysis must exist. This balance is achieved in a variety of ways. Cells have the ability to control protein synthesis by regulating transcription and translation (1, 2). Similarly, cells can also control protein degradation by using a variety of cellular processes such as lysosomal degradative pathways and the ubiquitin/proteasome pathway (3, 4).

The lysosomal degradative pathways can be separated into endocytosis, macroautophagy, crinophagy, pexophagy, micro-autophagy, and chaperone-mediated autophagy (CMA)1; only the latter pathway does not involve vesicular membrane traffic (5). Autophagy, literally "self-eating," is a cellular process that allows cells to remove proteins, organelles, and foreign bodies from the cytosol and deliver them to lysosomes for degradation.

CMA is a process activated during long term starvation in which cells selectively degrade proteins in order to recycle their amino acids or use them for energy. During nutrient deprivation, substrates that contain a consensus motif related to KFERQ (6) are recognized by a chaperone-cochaperone complex containing the heat shock cognate protein of 70 kDa (hsc70) (7, 8). Once this chaperone-cochaperone complex binds the substrate, it docks on the lysosomal membrane via a receptor known as the lysosomal associated membrane protein 2a (lamp2a) (9). The substrate then is unfolded (10), presumably by the chaperone-cochaperone complex, translocated into the lumen with the help of a lysosomal isoform of hsc70 (lyhsc70) (11), and degraded. Like most organelle protein import pathways, CMA is saturable as well as temperature-dependent (12, 13). The substrates for CMA also compete with one another for binding and import, which provides an experimental method for discovering new substrates (12). There have been several substrates identified for CMA including ribonuclease A (RNase A) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (14, 15). CMA has been reconstituted using lysosomes isolated from human fibroblasts and rat liver, which permits the study of CMA in a more mechanistic fashion (8, 16).

Ketone bodies are produced by the liver during long term starvation in response to rapid lipolysis. Ketone bodies can be utilized by muscle and contribute to the preservation of muscle mass during prolonged survival; they can also be used as an energy source for the brain (17, 18). Ketone bodies are comprised of three biologically active compounds, namely acetoacetate, {beta}-hydroxybutyrate (BOH), and acetone (19, 20). Interestingly, the increase in the concentration of circulating ketone bodies parallels the induction of CMA, which is also activated by prolonged starvation (5).

In this paper we demonstrate that treatment of cells with ketone bodies increases the proteolysis of long-lived proteins under conditions in which most proteolysis is due to CMA (28). We also show that the increase in proteolysis observed is at least in part due to the stimulation of CMA. Lastly, we show that ketone bodies induce CMA by oxidizing substrates, permitting them to be recognized by the CMA machinery and imported into the lysosome more efficiently.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Isolation of Lysosomes—Human embryonic fibroblasts (IMR-90) were obtained from Corriel Cell Repositories (Camden, NJ). Cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% newborn calf serum and 1% antibiotic (penicillin/streptomycin with fungicide; Invitrogen). IMR-90 cells were used for experiments at 20–40 population doubling levels (PDLs). To deprive cells of serum, cultures were washed several times with Hanks' balanced salts' solution (Invitrogen) and then placed in media without serum. Lysosomes from IMR-90 cells were isolated as described previously (21). Lysosomal matrix preparations were prepared by subjecting the lysosomes to hypotonic shock, as reported previously (22).

Lysosomal Latency Assay—To assess the integrity of the lysosomal membrane, we performed a {beta}-hexosaminidase latency study as described previously (13). Lysosomal preparations that contained >10% broken lysosomes, as determined by {beta}-hexosaminidase latency, were not used.

Chemicals and Antibodies—All chemicals were obtained from Sigma unless otherwise indicated. GAPDH was obtained from Roche Applied Science, and RNase A was obtained from Worthington Biochemical Company (Lakewood, NJ). Antibodies against lamp2a were obtained from Zymed Laboratories Inc.. Antibodies against GAPDH were obtained from Biodesign (Saco, ME), and antibodies against hsc70 were obtained from Maine Biotechnology Services (Portland, ME). Detection of the formation of carbonyl groups in proteins was performed by following the manufacturer's instructions using the OxyBlotTM oxidized protein detection kit supplied by Chemicon International (Temecula, CA). [14C]GAPDH and [14C]RNase A were radiolabeled by using [14C]formaldehyde reductive methylation as described previously (23).

Cellular Protein Degradation—Confluent cells were labeled with [3H]leucine (2 µCi/ml) for 48 h in media containing 10% newborn calf serum. Cells were washed twice with Hanks' balanced salt solution, and the media were replaced with either complete media or media without serum both containing excess (2.8 mM) unlabeled leucine (24). Aliquots of media (200 µl) were taken at the indicated time intervals and precipitated in 20% trichloroacetic acid. Proteolysis was determined by measuring the percent of acid soluble radioactivity compared with the total radioactivity of cell lysates prepared by the addition of 0.1 N NaOH and 0.1% sodium deoxycholate (24).

In Vitro Lysosomal Import and Protease Protection Assays— [14C]GAPDH and [14C]RNase A were incubated in the presence of isolated lysosomes for 1.5 h as described previously (13). Degradation was calculated as the percent of trichloroacetic acid-soluble radioactivity converted from the trichloroacetic acid-precipitable radioactivity (13). The protease protection assay was performed as described previously (7). Briefly, GAPDH was incubated with purified lysosomes treated with 100 µM chymostatin A. The GAPDH was incubated with the lysosomes for 30 min and then washed and treated with 10 µg of proteinase K and 1 µM CaCl2 for 15 min. The reaction was stopped by incubation with 4-(2-aminoethyl)-benzenesulfonyl fluoride. Lysosomes were solubilized and processed for SDS-PAGE.

Immunoprecipitation of GAPDH—Cells were placed in conditions of serum deprivation for 24 h. The cells were washed twice in ice-cold phosphate-buffered saline and incubated in Nonidet P-40 lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5 µg/ml phenylmethylsulfonyl fluoride, and 0.01 µg/ml each aprotinin, leupeptin, and pepstatin for 15 min The lysate was collected by centrifuging the samples at 14,000 x g for 15 min. Aliquots were then taken and precleared with protein A-Sepharose beads (Amersham Biosciences) for 1 h. Antibody (8 µg) was added to the precleared lysate and incubated overnight at 4 °C. The antibody-antigen complex was removed from the lysate by centrifuging the samples at 10,000 x g for 30 s to pellet the protein A-Sepharose beads. The supernatant was discarded, and beads washed an additional three times. The beads were resuspended in Laemmli sample buffer and subjected to SDS-PAGE followed by immunoblot analysis.

General Methods—Protein determination was performed using the Lowry method with bovine serum albumin as a standard (25). Protein detection methods such as SDS-PAGE (26) and immunoblotting (27) were visualized using chemiluminescence detection methods (Western Lightning; PerkinElmer Life Sciences). Radioactivity was determined by counting samples in a Packard liquid scintillation analyzer with quenching detected by an automatic external standard (Packard Tri-Carb 2100 TR). Densitometric analysis was performed by using Adobe Photoshop 7.0. Statistical analyses were preformed using the two-tailed Student's t test.



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  		FIG. 1.
Effect of BOH and acetoacetate on the degradation of total cellular protein. Protein degradation is calculated by labeling cellular protein and measuring the release of [3H]leucine over time and comparing the acid-soluble radioactivity to the total radioactivity, as described (24). a, MR-90 cells in the presence and absence of serum treated or not treated with 4 mM BOH. b, MR-90 cells in the presence and absence of serum treated or not treated with 4 mM acetoacetate. In panel a, time points from BOH-treated cells show statistically significant increased proteolysis under all conditions (p ≤ 0.001). In panel b, acetoacetate treatment at 4 and 26 h in the presence of serum shows statistically increased degradation compared with control (p ≤ 0.001).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In confluent cultures of IMR-90 human fibroblasts, serum withdrawal activates CMA (5, 28). Using pulse-chase analysis, we analyzed the effect of BOH on cellular proteolysis in IMR-90 cells maintained in media with or without serum (Fig. 1a). We observed an increase in proteolysis in BOH-treated cells maintained either with or without serum as compared with control. In cells maintained in media with serum, proteolysis increased to that of control cells maintained without serum. We found similar effects of BOH-induced proteolysis using Chinese hamster ovary cells (data not shown). Treating cells with acetoacetate resulted in an increase in proteolysis only in cells maintained in media containing serum (Fig. 1b). Because BOH had a more dramatic activation of CMA, we chose to focus on the BOH induction of CMA. Because CMA is activated by serum withdrawal, all cell cultures were serum-deprived 24 h prior to experimentation unless noted otherwise.

Treatment with oxidizing agents during prolonged nutrient deprivation may decrease lysosomal stability (29, 30). To address the issue of lysosomal stability we performed a {beta}-hexosaminidase latency assay. Lysosomes were isolated from cells in the absence or presence of BOH. The isolated lysosomes were kept at 37 °C, and the latency was measured at either 0, 15 or 30 min after isolation. The stability of the lysosomes from treated cells was indistinguishable from those isolated from treated cells at all time points (Fig. 2). After isolation, ~1% of the total lysosomes were broken in both treated and untreated lysosomes. After incubation at 37 °C for 15 and 30 min, the percentage of broken lysosomes rose to 1.5 ± 0.5 and 1.75 ± 0.5%, respectively. Therefore, we conclude that treatment with BOH does not affect the stability of the lysosomes.



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  		FIG. 2.
Stability of lysosomes isolated from untreated and BOH-treated IMR-90 cells. Lysosomes were isolated from IMR-90 cells maintained in media without serum, treated with or without 4 mM BOH, and subject to a latency test to determine stability (see "Materials and Methods"). Error bars represent S.D. of ±1; BOH-treated and control lysosomes were not significantly different.

 
To explore the mechanisms for the BOH-induced CMA, we performed in vitro CMA degradation assays using isolated lysosomes. In these assays, isolated lysosomes combined with substrates of CMA such as [14C]GAPDH and [14C]RNase A allowed us to determine the percentage of lysosomal protein degradation by measuring the change of trichloroacetic acid-precipitable radioactivity into trichloroacetic acid-soluble radioactivity (15, 28).

The lysosomes isolated from BOH-treated cells showed an increase in degradation of both [14C]GAPDH (Fig. 3a) and [14C]RNase A (Fig. 3b) as compared with controls. Degradation of both [14C]GAPDH and [14C]RNase increased with increased amounts of lysosomal protein (0,14, 28, and 56 µg) At 14 µg of lysosomal protein there was no significant difference in the degradation of [14C]GAPDH or [14C]RNase A between lysosomes isolated from control versus treated cells; however, at 28 and 56 µg of lysosomal protein from treated cells there was a 2- and 3-fold increase in the degradation of [14C]GAPDH, respectively, in treated compared with control cells. Similarly, with 28 and 56 µg of lysosomal protein there was a 3- and 5-fold increase in the degradation of [14C]RNase A, respectively, compared with control. These data indicate that the observed increase in proteolysis was due, at least in part, to the stimulation of CMA.

To determine the mechanism of activation of CMA by BOH we isolated lysosomes from cells and then added varying concentrations of BOH (Fig. 4). The degradation was enhanced by increasing the concentration of BOH, achieving a maximal response at 4 mM. The observed increase in the proteolysis of [14C]GAPDH and [14C]RNase A indicated that BOH was acting on the lysosome and/or substrates to elicit an effect.

We next wanted to determine whether BOH has an effect on the levels of key proteins involved in CMA, namely lamp2a and lyhsc70. The amount of lamp2a in the lysosome membrane is rate-limiting for CMA under a variety of physiological and pathological conditions (31). The amount of lyhsc70 can also be rate-limiting under conditions such as prolonged starvation (32). We isolated lysosomes from control and BOH-treated cells and measured protein levels by immunoblot analysis. We found that the amount of hsc70 and lamp2a in the membrane and lyhsc70 in the membrane and matrix of lysosomes were equivalent in control and treated cells (Fig. 5). Therefore, BOH was inducing CMA without altering the levels of these key proteins.



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  		FIG. 3.
The degradation of [14C]GAPDH and [14C]RNase A by isolated lysosomes. Lysosomes were isolated from cells maintained in media without serum and treated with (white bars) or without (black bars) 4 mM BOH. a, degradation of [14C]GAPDH was monitored by the conversion of acid-precipitable radioactivity into acid-soluble radioactivity (0 and 14 µg, not significant; *, p ≤ 0.001 for 28 and 50 µg). b, degradation of [14C]RNase A was monitored as for [14C]GAPDH (0 and 14 µg, not significant; *, p ≤ 0.001 for 28 and 50 µg).

 



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  		FIG. 4.
Degradation of [14C]GAPDH and [14C]RNase A by isolated lysosomes treated with varying concentrations of BOH. Lysosomes were isolated from IMR-90 cells maintained in media without serum and treated with increasing concentrations of BOH; n = 3 and 2 for each time point of GAPDH and RNase A, respectively. Error bars indicate S.D. of ±1 for GAPDH. The average and the range are shown for RNase A duplicates. All time points with intact lysosomes were statistically increased compared with broken lysosomes (p ≤ 0.001). Proteolysis is reported as percent of untreated lysosomes.

 
The proteolysis assays using isolated lysosomes (Figs. 3 and 4) reflect a combination of substrate binding, transport, and degradation (13). To establish whether substrate was entering BOH-treated lysosomes more efficiently than untreated lysosomes, we performed a proteinase K protection assay. We isolated lysosomes from IMR-90 cells and treated them with protease inhibitors. Lysosomes were then incubated with BOH and GAPDH or GAPDH alone. After 1 h of incubation, we washed the lysosomes and treated with proteinase K to cleave any remaining substrate bound to the lysosomal membrane. We then carried out immunoblots of the isolated lysosomes to measure the amount of GAPDH that had been transported into the lysosomes (Fig. 6). The lysosomes incubated with BOH showed a 2.5-fold increase in the amount of GAPDH that was transported into the lysosome (Fig. 6). These data suggest that in a reaction mixture containing BOH, GAPDH, and lysosomes, the GAPDH undergoes a more efficient uptake compared with a reaction mixture deprived of BOH, suggesting that BOH acts on the substrate, the lysosome, or both.

Next, we isolated lysosomes from IMR-90 cells. The lysosomes and/or GAPDH/RNase A were pretreated with 4 mM BOH and then subjected to a transport assay (Fig. 7). We found that when substrate was preincubated with BOH there was a 2-fold increase in its degradation compared with untreated substrates. BOH-pretreated lysosomes degraded both substrates at the same rate as untreated lysosomes. These data suggest that BOH acts on substrates and causes them to be degraded by CMA more efficiently.



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  		FIG. 5.
Protein levels of lyhsc70 and lamp2a in cells treated with or without BOH. Lysosomes (Ly) were isolated from IMR-90 cells maintained in media without serum, treated with (+) or without (-) BOH, and subjected to immunoblot. Protein (100 µg) was loaded per well. Bands were quantitated using scanning densitometry. There was no statistical difference between lamp2a or hsc70 in the treated or untreated groups. Mtx, matrix; Mem, membrane. These results are representative of three different experiments.

 



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  		FIG. 6.
Analysis of GAPDH uptake into lysosomes of BOH-treated and untreated cells. Lysosomes were isolated from IMR-90 cells maintained in media without serum and treated (+) or not treated (-) with BOH. Shown is a Western blot analysis of lysosomes subjected to a GAPDH/proteinase K protection assay.

 



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  		FIG. 7.
Preincubation of substrates, but not lysosomes, with BOH increases degradation. Substrate (sample set 3) or lysosomes (sample set 2) were preincubated with 4 mM BOH for 20 min prior to transport assay. Control lysosomes were untreated with BOH (sample set 4) or contained only substrate (sample set 1); n = 3 for all experiments. Error bars indicate S.D. of ±1. The increased proteolysis in sample set 3 is statistically significant (p ≤ 0.001) for both GAPDH and RNase A. Values for sample sets 2 and 4 were not significantly different for either GAPDH or RNase A.

 
One possibility is that BOH directly or indirectly causes proteins to be oxidized, making the damaged proteins better substrates for CMA. To test this idea, we looked at the accumulation of oxidized GAPDH in BOH-treated IMR-90 cells. We immunoprecipitated GAPDH from either BOH-treated or untreated cells and then performed an OxyBlotTM assay, which derivatizes carbonyl groups to a 2,4-dinitrophenylhydrazone (DNP) moiety. The DNP moiety can then be detected using anti-DNP antibodies and is a method to assay for one form of oxidative damage to a protein. We then subjected the immunoprecipitated GAPDH to an OxyBlotTM assay followed by SDS-PAGE and immunoblot analysis using the anti-DNP antibody as our primary antibody. We found that GAPDH from BOH-treated cells was oxidized ~3-fold more than GAPDH from control cells, suggesting one mechanism for the stimulation of CMA by BOH (Fig. 8a,). RNase A is not an intracellular protein, so we could not follow the same procedures as for GAPDH. In this case, we incubated RNase A with 4 mM BOH and found that there was an increase in the appearance of carbonyl groups compared with control. RNase A incubated with BOH for 4 or 24 h showed ~2- and 3-fold increases, respectively, in carbonyl groups compared with untreated RNase A (Fig. 8b).

Finally, to determine whether the stimulation of CMA by BOH was specific for BOH, we tested compounds structurally similar to BOH, butanone, and butanol. We treated cells with both butanone and butanol (4 mM each) (Fig. 9a), but we did not observe any effect on proteolysis in the absence or presence of serum. We next asked if another compound whose concentration increases during starvation, glycerol, could stimulate CMA. We treated cells with glycerol concentrations ranging from 0.05 to 5.0 mM and measured their affects on CMA (Fig. 9b). There was no significant affect of glycerol on the stimulation of proteolysis. Taken together, these data suggest that BOH stimulates CMA in a specific manner.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Five lines of evidence indicate that ketone body formation leads to increased proteolysis by CMA. First, IMR-90 cells treated with BOH and, to a lesser extent, acetoacetate increased proteolysis in cells maintained in serum-supplemented media (Fig. 1, a and b). Acetone could not be tested because of its volatility.2 Second, we found that lysosomes isolated from BOH-treated cells transported and degraded substrates of CMA at a higher rate than control lysosomes (Fig. 3, a and 3b). Third, BOH-treated substrates were degraded by lysosomes at a higher rate than untreated substrates, suggesting that BOH acts through substrate modification to stimulate CMA (Fig. 7). Fourth, BOH incubation with RNase A increases the formation of carbonyl groups in RNase A (Fig. 8b). Lastly, we demonstrated that GAPDH, a substrate for CMA, immunoprecipitated from cells treated with BOH showing a higher occurrence of oxidative damage as compared with control (Fig. 8a).



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  		FIG. 8.
OxyBlotTM analysis of GAPDH and RNase A. a, GAPDH was immunoprecipitated using anti-GAPDH antibody from cells maintained in media without serum and supplemented with (+) or without (-) 4 mM BOH for 24 h. b, RNase A was incubated with BOH for either 4 or 24 h and subjected to OxyBlotTM analysis followed by SDS-PAGE and immunoblotting.

 



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  		FIG. 9.
Effects of butanone, butanol, and glycerol on total cellular proteolysis. a, IMR-90 cells treated with compounds similar to BOH, 4 mM butanone and 4 mM butanol, showed no effect on proteolysis. b, similarly, 5 mM glycerol had no effect on total cellular proteolysis; n = 3 for all time points. Error bars indicate S.D. of ±1. Differences in time points are not statistically significant (p > 0.05).

 
The effects of ketone bodies have been extensively studied on tissues that utilize them for energy, such as skeletal muscle and brain. The use of ketone bodies as an energy source prevents the catabolism of essential proteins and preserves amino acid pools within the cell during times of nutritional stress (33–36). The effects of ketone bodies on cell types that do not utilize ketone bodies for energy, such as IMR-90 cells, have not been closely studied even though they survive in an environment in which ketone bodies reach significantly high levels of 1–12 mM (37). The ability of BOH to increase protein breakdown in times of nutritional stress may be one more evolutionary mechanism for cells to survive prolonged nutrient deprivation without using ketone bodies for energy.

Previous studies have shown that oxidized proteins make better substrates for CMA (38). Our results are consistent with those of Kiffin et al. (38), who demonstrated that oxidized proteins have the ability to bind and translocate into the lysosome more rapidly than non-oxidized proteins. The mechanism of enhanced substrate uptake due to oxidation is not clear. We do know that substrates must be unfolded prior to translocating across the lysosomal membrane (10). Therefore, the unfolding of proteins after oxidation may promote recognition by the chaperone-cochaperone complex and increase the rate of delivery and uptake of oxidized substrates into the lysosome.

It is interesting to note that in Fig. 4 the broken lysosomes actually have a decreasing degradative capacity in the presence of increasing concentrations of BOH. This may be explained by the fact that increasing concentrations of BOH may be damaging and inactivating the degradative enzymes, thus causing less proteolysis. Future studies could focus on the effect that BOH treatment has on enzyme function. Also in Fig. 3 we observed that there was no difference between the amount of degradation between 0 and 14 µg of lysosomal protein. One explanation of this observation is that our degradation assay is not sensitive enough to detect changes in degradation with this amount of lysosomal protein. With no lysosomes added we found 1–2% protein degradation. Undoubtedly, one needs enough of a signal above 1–2% to reflect CMA. If this were the case one would expect to see an increase in degradation by adding higher concentrations of lysosomes, which is in fact what we observed.

We demonstrated that there was an increase in the degradation of [14C]GAPDH by lysosomes isolated from cells treated with 4 mM BOH for 24 h (Fig. 3). However, we saw no increase in proteolysis when lysosomes were preincubated with 4 mM BOH for 20 min (Fig. 7). One explanation for this apparent discrepancy is that isolated lysosomes normally have CMA substrates bound. These substrates will compete with [14C]GAPDH or [14C]RNase for uptake. The lysosomes isolated from BOH-treated cells will have fewer CMA substrates bound because they are more efficiently translocated and degraded (Fig. 6). Therefore, lysosomes isolated from BOH-treated cells will have a higher activity for added radiolabeled substrates. The lysosomes preincubated with BOH for 20 min may not have had sufficient time to process nonradioactive bound CMA substrates. We are unable to test this idea, because the isolated lysosomes are not stable during prolonged incubation times.

Whenever we compared intracellular proteolysis rates, cells were of equivalent PDLs between 20 and 40. However, proteolytic rates between experiments were somewhat variable (Figs. 1, a and b and 9, a and b). In 100 independent experiments we obtained half-lives of 48 ± 6 and 28 ± 6 h in cells maintained with or without serum, respectively. IMR-90 cells in media without serum have been shown to increase the half-life of long-lived proteins by 20 min per PDL (38). Therefore, varying PDLs between 20 and 40 h will lead to variability in a t1/2 of ±7 h.

There are several possible mechanisms to explain BOH stimulation of CMA. First, BOH likely causes protein oxidation by increasing reactive oxygen species, which could lead to an increase in total oxidized proteins (39). Because 30% of cytosolic proteins are substrates for CMA, this could result in higher rate of degradation for these proteins by CMA. Kiffin et al. (38) demonstrated an increase in the levels of several CMA components, such as lamp2a, in response to oxidation-induced activation of CMA (38). These results are somewhat different from ours, as we could not see a measurable change in the levels of lamp2a or the luminal chaperone. These differences could be explained by the fact that Kiffin et al. (38) dosed their rodents twice with paraquat or hydrogen peroxide to oxidize proteins. Treatment with pro-oxidants of this magnitude may increase the levels of oxidized proteins and force the cell to up-regulate components of CMA in order to deal with the increased demand of substrates. BOH, on the other hand, may only oxidize a small percentage of proteins, permitting the cell to degrade the damaged proteins without increasing components of the pathway.

Second, BOH, a consumer of NAD+ (40), may elevate the NADH/NAD+ ratio, similar to what occurs during starvation (41), and cause the cell to activate CMA as part of the nutrient stress response. The cells would induce CMA without the need to up-regulate components of the pathway. This does not seem likely, because immunoprecipitated GAPDH from BOH-treated cells had a 3-fold increase in the level of oxidation as compared with control. Future studies should investigate the effects of oxidants of varying strengths on the modification of substrates and the subsequent activation of CMA.

Third, several studies suggest that ketone bodies have the ability to cause increased free radical formation and lipid peroxidation in erythrocytes and endothelial cells (42, 43). We did observe an increase in proteolysis in acetoacetate-treated cells maintained in serum, but not in cells maintained without serum (Fig. 1b). This could be explained by the fact that acetoacetate has the ability to down-regulate the insulin receptor at the mRNA level by 56% (44). Down-regulation of the insulin receptor would stimulate the cell to respond to nutrient deprivation and activate CMA. This is one explanation as to why we only see an activation of CMA in cells maintained in serum. Future studies should look at what other factors contribute to CMA activation.

In these studies we have demonstrated that ketone bodies, more specifically BOH, stimulate CMA by causing the oxidation of substrates. In addition, during prolonged starvation CMA is activated because of increased lamp2a in the lysosomal membrane and increased lyhsc70 in the lysosomal lumen (9, 31). Our data indicate that ketone bodies can also stimulate CMA by affecting substrate proteins during prolonged starvation in vivo. This finding gives us further insight into the physiological importance of CMA stimulation during times of nutrient deprivation.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Molecular and Cellular Physiology, Tufts University School of Medicine, Arnold 809, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-0408; Fax: 617-636-0445; E-mail: Patrick.Finn{at}tufts.edu.

1 The abbreviations used are: CMA, chaperone-mediated autophagy; BOH, {beta}-hydroxybutyrate; DNP, 2,4,dinitrophenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hsc70, 70-kDa heat shock cognate protein; lamp2a, lysosomal-associated membrane protein 2a; lyhsc70, 70-kDa lysosomal heat shock cognate protein; PDL, population doubling level. Back

2 P. F. Finn and J. F. Dice, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Ana Maria Cuervo, Dr. Ira Herman, Dr. Laura Liscum, and Nicholas T. Mesires, M.S. for valuable discussions and critical evaluation of this manuscript. We also thank Dr. Alfred J. Meijer for suggesting to us that ketone bodies may activate CMA.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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