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).
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 and9, 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 t
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.
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@tufts.edu .
1 The abbreviations used are: CMA, chaperone-mediated autophagy; BOH, 
-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.
2 P. F. Finn and J. F. Dice, unpublished results.
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.
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