Abstract

Mitochondria integrate metabolic networks for maintaining bioenergetic requirements. Deregulation of mitochondrial metabolic networks can lead to mitochondrial dysfunction, which is a common hallmark of many diseases. Reversible post-translational protein acetylation modifications are emerging as critical regulators of mitochondrial function and form a direct link between metabolism and protein function, via the metabolic intermediate acetyl-CoA. Sirtuins catalyze protein deacetylation, but how mitochondrial acetylation is determined is unclear. We report here a mechanism that explains mitochondrial protein acetylation dynamics in vivo. Food withdrawal in mice induces a rapid increase in hepatic protein acetylation. Furthermore, using a novel LC–MS/MS method, we were able to quantify protein acetylation in human fibroblasts. We demonstrate that inducing fatty acid oxidation in fibroblasts increases protein acetylation. Furthermore, we show by using radioactively labeled palmitate that fatty acids are a direct source for mitochondrial protein acetylation. Intriguingly, in a mouse model that resembles human very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency, we demonstrate that upon food-withdrawal, hepatic protein hyperacetylation is absent. This indicates that functional fatty acid oxidation is necessary for protein acetylation to occur in the liver upon food withdrawal. Furthermore, we now demonstrate that protein acetylation is abundant in human liver peroxisomes, an organelle where acetyl-CoA is solely generated by fatty acid oxidation. Our findings provide a mechanism for metabolic control of protein acetylation, which provides insight into the pathophysiogical role of protein acetylation dynamics in fatty acid oxidation disorders and other metabolic diseases associated with mitochondrial dysfunction.

INTRODUCTION

Post-translational modifications (PTMs) arm the cell with a highly dynamic mechanism for regulation of cellular pathways. One of the frequently modified residues is lysine, which is targeted by a variety of PTMs, including ubiquitination, methylation, biotinylation and acetylation. Among these PTMs, lysine acetylation has proven to be a major regulatory mechanism of metabolic pathways (1–3). Acetylation is a reversible modification which involves the transfer of an acetyl group to ɛ-amino group of lysines. It was first characterized on histones and has been known for decades as an essential regulatory mechanism of chromatin dynamics (4). Recently, numerous large-scale proteomic studies have identified abundant lysine acetylation on proteins located particularly in mitochondria and on metabolic enzymes in general (4–7). Functional studies have also shown that mitochondrial enzymes are regulated by acetylation (3,8–12). Among these are enzymes in the urea cycle, TCA cycle, fatty acid oxidation and ketogenesis (3,8–12). The physiological significance of lysine acetylation is further underscored by its evolutionary conservation from bacteria to humans (13,14).

Schwer et al. (15) were the first to report that mitochondrial lysine acetylation can globally change with metabolic conditions. In livers of mice subjected to caloric restriction (CR), lysine acetylation increased as compared with mice fed a normal diet (15). In other studies it was shown that high-fat diet feeding and chronic ethanol consumption also resulted in global hepatic lysine hyperacetylation (16,17). Furthermore, recent quantitative large-scale proteomics demonstrated that upon CR indeed the majority of differentially acetylated proteins have increased acetylation (6). These findings suggest there is a link between the metabolic state of the cell and the level of protein acetylation.

The acetylation state of mitochondrial proteins is controlled by the mitochondrial deacetylase SIRT3, which removes acetyl groups from lysines in a NAD+-dependent manner. SIRT3 is a member of the sirtuin family and one of the three sirtuins localized in the mitochondria, the other two being SIRT4 and SIRT5 (18). From the three mitochondrial sirtuins, SIRT3 is the only one to possess robust deacetylase activity on multiple proteins (6,8,10–12,19). On the contrary to deacetylation, little is known about how lysine acetylation is catalyzed in mitochondria. Recent studies have indicated that GCN5L1 might play a role in mitochondrial lysine acetylation (20), but additional components might be necessary for regulating lysine acetylation as well. The fact that mitochondrial metabolism generates large amounts of acetyl-CoA have led to speculations that mitochondrial acetylation can occur non-enzymatically (21–25). Indeed, in vitro experiments in cell lysates have suggested that the chemical conditions in mitochondria are favorable for non-enzymatic mitochondrial lysine acetylation (26). However, in vivo evidence for a non-enzymatic lysine acetylation mechanism in mammals is lacking.

We have now uncovered a mechanism that explains mitochondrial lysine acetylation dynamics in mouse liver and human cells. We provide in vivo evidence that liver hyperacetylation directly relies on fatty acid oxidation flux. Mice deficient in fatty acid oxidation are unable to increase acetylation upon fasting and by using radioactively labeled palmitate, we show that fatty acids are the direct source of acetyl units for lysine acetylation in the mitochondria. Furthermore, we are the first to demonstrate that lysine acetylation is abundant in human liver peroxisomes, an organelle where acetyl-CoA is solely generated by fatty acid oxidation.

RESULTS

Hepatic protein acetylation increases in response to food withdrawal

To adapt to periods of nutrient deprivation, the liver switches from carbohydrate utilization and lipogenesis to fatty acid oxidation and ketone body production (27). To evaluate how this metabolic switch influences hepatic protein acetylation, we studied acetylation dynamics in the mouse liver during fasting. For this end, we fasted mice for up to 16 h. To monitor the fasting response we measured glucose and ketone body (3-hydroxybutyrate) levels in plasma at different time intervals during food withdrawal. Blood glucose levels declined rapidly in the first 6 h of fasting from 11 to 7 mmol/l and continued to gradually decrease throughout the fasting period down to 5 mmol/l (Fig. 1A). The levels of 3-hydroxybutyrate in plasma started to increase after 4 h of fasting to 0.2 mmol/l and further increased during the period of fasting to 1.4 mmol/l after 16 h (Fig. 1B), indicating increased rates of hepatic fatty acid oxidation. Interestingly, hepatic acetylation globally increased during the period of food withdrawal. Protein acetylation increased 2.4-fold already 4 h after food withdrawal, which coincided with the first observed elevation of plasma ketone body levels. After 16 h of food withdrawal, hepatic protein acetylation had increased even further up to a total of 3-fold induction upon food withdrawal (Fig. 1C).

Figure 1.

Hepatic protein acetylation increases in response to food withdrawal. (AC) Mice were subjected to food withdrawal for up to 16 h. Levels of blood glucose (A); plasma ketone bodies (3-hydroxybutyrate) (B); and hepatic protein acetylation (C) were measured at different time intervals.

Figure 1.

Hepatic protein acetylation increases in response to food withdrawal. (AC) Mice were subjected to food withdrawal for up to 16 h. Levels of blood glucose (A); plasma ketone bodies (3-hydroxybutyrate) (B); and hepatic protein acetylation (C) were measured at different time intervals.

Quantitative analysis of protein acetyl lysine

Next, we wanted to study protein acetylation dynamics in human cells. For this we set-up an alternative methodology to analyze quantitative lysine acetylation, that does not rely on the use of antibodies. This method is based on digesting proteins or cell lysates with pronase to yield free amino acids, followed by analysis of lysine and acetyl lysine by LC–MS/MS. Previously, similar methods were used to identify histone acylation dynamics (28). We validated our method by spiking samples with N(ɛ)-acetyl lysine and by chemically acetylating purified glutamate dehydrogenase (GDH) followed by western blot analysis or LC–MS/MS analysis. Incubation of purified GDH with pronase at 37°C O/N resulted in formation of lysine in a reproducible manner (Fig. 2A). Incubation with pronase alone did not produce significant amounts of lysine, indicating that auto-proteolysis of pronase to single lysine did not contribute to a large extent to the observed lysines that were released by digesting GDH. Furthermore, spiking the samples with either 0.1 or 1 nmol acetyl lysine resulted in a recovery of 80–100% of added acetyl lysine (Fig. 2B), demonstrating that acetyl lysine is stable during pronase incubation, acetyl lysine extraction and LC–MS/MS analysis. An intriguing aspect of our analysis is that we are now able to provide stoichiometric data on acetyl lysine over lysine levels in single proteins and in lysates. In purified bovine GDH 1 out of 500 lysines was acetylated (Fig. 2A and B).

Figure 2.

Quantitative measurements of acetylated lysines. (A and B) Purified bovine GDH was incubated with and without pronase and addition of acetyl lysine (AcK) spikes at 0.1 and 1 nmol, followed by lysine analysis (A) and acetyl lysine analysis (B). (C) GDH was acetylated in vitro using increasing concentrations (0–10 mm) of acetic anhydride followed by analysis of lysine acetylation by western blot. GDH was detected by coomassie staining. (D) Quantification of acetyl lysine signals from C. (E) Acetyl lysine/lysine ratios (AcK/total K) were analyzed by LC–MS/MS after pronase incubation. (F) Correlation of western blot acetylation analysis and LC–MS/MS analysis.

Figure 2.

Quantitative measurements of acetylated lysines. (A and B) Purified bovine GDH was incubated with and without pronase and addition of acetyl lysine (AcK) spikes at 0.1 and 1 nmol, followed by lysine analysis (A) and acetyl lysine analysis (B). (C) GDH was acetylated in vitro using increasing concentrations (0–10 mm) of acetic anhydride followed by analysis of lysine acetylation by western blot. GDH was detected by coomassie staining. (D) Quantification of acetyl lysine signals from C. (E) Acetyl lysine/lysine ratios (AcK/total K) were analyzed by LC–MS/MS after pronase incubation. (F) Correlation of western blot acetylation analysis and LC–MS/MS analysis.

To demonstrate the quantitative nature of our method we first chemically acetylated GDH (Fig. 2C). Quantitating acetyl lysine signals from the western blot showed that incubating GDH with acetic anhydride resulted in a gradual increase of acetylation when using up to 5 mm acetic anhydride (Fig. 2D). Incubating with 10 mm acetic anhydride did not increase acetylation any further (Fig. 2D). Analyzing chemically acetylated GDH by pronase incubation and LC–MS/MS analysis, demonstrated a similar acetylation increase (Fig. 2E). Comparing analysis of acetylation levels by western blot with analysis of acetylation levels by LC–MS/MS analysis gave a linear correlation (R2 = 0.97). Thus, our method reliably analyzes quantitative lysine acetylation, which is suited for analysis of quantitative global lysine acetylation in human cells.

Acetylation increases in fibroblasts with activated fatty acid oxidation

We quantified acetylated lysines in human fibroblasts cultured in low glucose medium containing palmitate and carnitine or in regular high glucose medium for 96 h. Incubating fibroblasts in low glucose medium containing fatty acids and carnitine, induces a metabolic state characterized by high fatty acid oxidation flux (29). We analyzed protein-derived lysine (Fig. 3A) and acetyl lysine (Fig. 3B) in five independent human fibroblasts cell lines. Lysine levels were not significantly different between Dulbecco's modified Eagle medium (DMEM) and minimal essential medium (MEM)/palmitate conditions (Fig. 3A), whereas acetyl lysine levels were significantly higher in MEM/palmitate conditions (Fig. 3B). Therefore, the acetyl lysine/lysine ratio was higher in MEM/palmitate conditions as compared with DMEM (Fig. 3C). This indicated that MEM/palmitate conditions induced global lysine acetylation levels.

Figure 3.

Acetylation increases in fibroblasts with activated fatty acid oxidation. Five human dermal fibroblast cell lines were used to analyze lysine acetylation upon culturing cells in either rich DMEM containing high glucose (25 mm) and 10% FBS (white bars: DMEM) or in palmitate/carnitine containing MEM with low glucose (5 mm) and without FBS (black bars: MEM + palmitate). (A) Protein-derived lysine levels in pronase treated cell lysates analyzed by LC–MS/MS (mean ± SEM, n = 5). (B) Acetyl lysine levels in pronase treated cell lysates analyzed by LC–MS/MS (mean ± SEM, n = 5 and *** indicates P < 0.001). (C) Ratio of acetyl lysine/lysine derived from the data presented in (A) and (B) (mean ± SEM, n = 5 and *** indicates P < 0.001).

Figure 3.

Acetylation increases in fibroblasts with activated fatty acid oxidation. Five human dermal fibroblast cell lines were used to analyze lysine acetylation upon culturing cells in either rich DMEM containing high glucose (25 mm) and 10% FBS (white bars: DMEM) or in palmitate/carnitine containing MEM with low glucose (5 mm) and without FBS (black bars: MEM + palmitate). (A) Protein-derived lysine levels in pronase treated cell lysates analyzed by LC–MS/MS (mean ± SEM, n = 5). (B) Acetyl lysine levels in pronase treated cell lysates analyzed by LC–MS/MS (mean ± SEM, n = 5 and *** indicates P < 0.001). (C) Ratio of acetyl lysine/lysine derived from the data presented in (A) and (B) (mean ± SEM, n = 5 and *** indicates P < 0.001).

Protein acetylation is abundant in mitochondria and peroxisomes

Since most experiments on in vivo lysine acetylation are performed in mice and relatively little is known about lysine acetylation in human tissue, we wanted to verify lysine acetylation distribution in organelles of human liver. To obtain detailed information on organellar distribution of protein acetylation outside of the nucleus in human tissue, we generated a post-nuclear supernatant (PNS) from human liver and separated peroxisomes and mitochondria from ER, lysosomes and cytosol. Using antibodies against catalase, electron transfer flavoprotein (ETF), endoplasmic reticulum protein 72 (ERp72) and lysosomal-associated membrane protein 1 (LAMP1), clear separation of organelles was verified (Fig. 4A). We observed abundant acetylation of proteins in fractions containing peroxisomes and mitochondria, whereas acetylation was much lower in ER/lysosomal and cytosolic protein fractions (Fig. 4B). These differences could not be accounted for by differences in protein loading, because equal amounts of protein were loaded (Fig. 4C).

Figure 4.

Acetylation is abundant in mitochondria and peroxisomes in human liver. Western blot analysis of peroxisomal, mitochondrial, ER-lysosomal and cytosolic fractions of human liver obtained by fractionation of post-nuclear supernatants using nycodenz gradient centrifugation. (A) Catalase (peroxisomes), ETF (mitochondria), ERp72 (ER) and LAMP1 (lysosomes) were used as organellar protein marker. Equal amounts of protein were loaded in each lane. (B) Lysine acetylation levels in different organelles of human liver analyzed with α-acetyl lysine western blot. Equal amounts of protein were loaded in each lane. (C) Coomassie staining of samples in (A) and (B).

Figure 4.

Acetylation is abundant in mitochondria and peroxisomes in human liver. Western blot analysis of peroxisomal, mitochondrial, ER-lysosomal and cytosolic fractions of human liver obtained by fractionation of post-nuclear supernatants using nycodenz gradient centrifugation. (A) Catalase (peroxisomes), ETF (mitochondria), ERp72 (ER) and LAMP1 (lysosomes) were used as organellar protein marker. Equal amounts of protein were loaded in each lane. (B) Lysine acetylation levels in different organelles of human liver analyzed with α-acetyl lysine western blot. Equal amounts of protein were loaded in each lane. (C) Coomassie staining of samples in (A) and (B).

Interestingly, mitochondria and peroxisomes are metabolically active organelles and both are characterized by a high capacity for fatty acid oxidation. Combined with our observation that lysine acetylation seems to be correlated with a metabolic condition characterized by high rates of fatty acid oxidation, we hypothesized that levels of protein acetylation might be correlated with the capacity of an organelle to oxidize fatty acids.

Acetyl-CoA generated by fatty acid oxidation directly provides acetyl units for mitochondrial protein acetylation

To determine whether the observed hyperacetylation during fasting indeed relies on acetyl-CoA generated by fatty acid oxidation, we set-up a cell model in which we could track acetyl units generated from fatty acids to protein acetyl groups. To induce a metabolic state in cells that is characterized by high fatty acid oxidation rate, Fao liver cells were cultured in fatty acid and carnitine-rich medium. To evaluate our model conditions we compared palmitate oxidation in cells cultured either in glucose-rich or fatty acid-rich medium. As expected, in fatty acid-rich medium the cells exhibited a high rate of palmitate oxidation, whereas in glucose-rich medium, palmitate oxidation was almost absent (Fig. 5A). Because previously Wellen et al. (30) demonstrated that acetyl units derived from glucose can be a source for nuclear histone acetylation, we wanted to verify this in our model system. Indeed, H3K9 and H4K8 were more acetylated in glucose-rich medium than in fatty acid-rich medium (Fig. 5B).

Figure 5.

Acetyl-CoA generated by fatty acid oxidation directly provides acetyl units for mitochondrial protein acetylation. (A) Palmitate oxidation rate in Fao hepatoma cells subjected to glucose-rich and fatty acid-rich culture conditions. Glucose-rich medium contained 5.6 mm glucose and dialyzed serum, whereas medium for fatty acid-rich conditions contained 5.6 mm galactose, 50 µm carnitine, 100 µm palmitate and dialyzed serum. The rate was assessed by measuring 3H2O generated by oxidation of [9,10-3H]-palmitate (mean ± SEM, n = 3 and ** indicates P < 0.01). (B) Acetylation levels of H3K9 and H4K8 in Fao cells in glucose-rich and fatty acid-rich conditions. (C) Graphical summary of the 14C-labeled mitochondrial protein acetylation generated through 14C-palmitate oxidation and experimental set-up. Fao cells were grown in fatty acid-rich conditions in medium containing 14C-palmitate, with and without carnitine supplementation for 18 h. Acetylated proteins were purified using α-acetyl lysine immunoprecipitation, resolved by SDS-PAGE and 14C incorporation into acetylated proteins was analyzed using radiography. (D) Distribution of cytosolic marker LDH and mitochondrial marker GDH in organellar fractions after fractionation of Fao cells by differential centrifugation. (E) Palmitate oxidation rate in Fao cells with and without carnitine supplementation. The rate was assessed by quantifying 14C incorporation into CO2 (mean ± SEM, n = 4 and ** indicates P < 0.01). (F and G) Radiography of 14C incorporated into total cell lysates (F) and acetylated proteins (G) in mitochondrial and cytosolic fractions from cells exposed to 14C-palmitate with and without carnitine supplementation.

Figure 5.

Acetyl-CoA generated by fatty acid oxidation directly provides acetyl units for mitochondrial protein acetylation. (A) Palmitate oxidation rate in Fao hepatoma cells subjected to glucose-rich and fatty acid-rich culture conditions. Glucose-rich medium contained 5.6 mm glucose and dialyzed serum, whereas medium for fatty acid-rich conditions contained 5.6 mm galactose, 50 µm carnitine, 100 µm palmitate and dialyzed serum. The rate was assessed by measuring 3H2O generated by oxidation of [9,10-3H]-palmitate (mean ± SEM, n = 3 and ** indicates P < 0.01). (B) Acetylation levels of H3K9 and H4K8 in Fao cells in glucose-rich and fatty acid-rich conditions. (C) Graphical summary of the 14C-labeled mitochondrial protein acetylation generated through 14C-palmitate oxidation and experimental set-up. Fao cells were grown in fatty acid-rich conditions in medium containing 14C-palmitate, with and without carnitine supplementation for 18 h. Acetylated proteins were purified using α-acetyl lysine immunoprecipitation, resolved by SDS-PAGE and 14C incorporation into acetylated proteins was analyzed using radiography. (D) Distribution of cytosolic marker LDH and mitochondrial marker GDH in organellar fractions after fractionation of Fao cells by differential centrifugation. (E) Palmitate oxidation rate in Fao cells with and without carnitine supplementation. The rate was assessed by quantifying 14C incorporation into CO2 (mean ± SEM, n = 4 and ** indicates P < 0.01). (F and G) Radiography of 14C incorporated into total cell lysates (F) and acetylated proteins (G) in mitochondrial and cytosolic fractions from cells exposed to 14C-palmitate with and without carnitine supplementation.

To test our hypothesis that fatty acids directly contribute acetyl units for acetylation of mitochondrial proteins, we exposed cells to 14C-labeled palmitate in carnitine-depleted and carnitine-rich medium (Fig. 5C). Next, we isolated mitochondrial and cytosolic fractions, purified acetylated proteins by immunoprecipitation using antibodies against acetylated lysines and analyzed 14C-label incorporation into acetyl groups of proteins by autoradiography (Fig. 5C). To verify the subcellular fractionation we measured activities of marker enzymes GDH and lactate dehydrogenase (LDH) for mitochondria and cytosol respectively. Using differential centrifugation, we obtained significantly enriched mitochondrial and cytosolic fractions with <10% contamination of mitochondrial fraction with cytosol and vice versa (Fig. 5D). Because we used carnitine-depleted medium, the oxidation rate of palmitate was significantly increased during the 18 h of incubation when carnitine was supplemented to the cells (Fig. 5E). We loaded both the total cell lysate and purified acetylated proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and analyzed 14C label incorporation with autoradiography. We observed that incorporation of 14C label into mitochondrial and cytosolic proteins from total cell lysate directly depended on carnitine supplementation and thus on palmitate oxidation, as it was considerably lower in cells cultured in carnitine-depleted medium (Fig. 5F). To analyze palmitate driven acetylation we looked at autoradiography of purified acetylated proteins (Fig. 5G). Interestingly, label incorporation into acetylated proteins was considerably higher in mitochondrial fractions in comparison to cytosolic fractions and it was also carnitine dependent (Fig. 5G). This demonstrates that acetyl-CoA produced from palmitate oxidation can be directly used for protein acetylation and that palmitate derived acetylation is to great extent mitochondrial.

Fatty acid oxidation is essential for hepatic hyperacetylation in mice

Our in vitro experiments demonstrate that acetyl-CoA generated by fatty acid oxidation serves as a source of mitochondrial protein acetylation and therefore we reasoned that fatty acid oxidation flux could be a determining factor of mitochondrial protein acetylation levels. To examine this in vivo, we investigated acetylation dynamics in mouse models with defective fatty acid oxidation. First we analyzed acetylation in the livers of mice lacking peroxisome proliferator-activated receptor alpha (PPARα). PPARα is essential for the metabolic switch that takes place during fasting as it induces amongst others the expression of genes involved in mitochondrial and peroxisomal fatty acid oxidation in response to high lipid influx into the liver. To activate PPARα and thereby mimic the fasting response we used its agonist WY14643 (31,32). Liver lysates from mice that were fed a diet with WY14643 showed high levels of protein acetylation as compared with liver lysates from mice that were fed a control diet (Fig. 6A). Notably, administration of WY14643 to PPARα knock-out (KO) mice did not result in increased protein acetylation (Fig. 6A), demonstrating that increased acetylation levels are indeed dependent on PPARα activation.

Figure 6.

Hepatic protein hyperacetylation does not occur in mice deficient in fatty acid oxidation. (A) Protein acetylation levels in liver lysates of WT and PPARα KO mice fed a diet with WY14643 for 2 weeks or a control diet. (B) Protein acetylation levels in whole liver lysates of WT and LCAD KO mice which were fed or fasted overnight. (C) SIRT3 protein expression in the livers of WT and LCAD KO mice, which were fed or fasted overnight.

Figure 6.

Hepatic protein hyperacetylation does not occur in mice deficient in fatty acid oxidation. (A) Protein acetylation levels in liver lysates of WT and PPARα KO mice fed a diet with WY14643 for 2 weeks or a control diet. (B) Protein acetylation levels in whole liver lysates of WT and LCAD KO mice which were fed or fasted overnight. (C) SIRT3 protein expression in the livers of WT and LCAD KO mice, which were fed or fasted overnight.

To directly link increased acetylation levels during fasting to fatty acid oxidation, we studied acetylation levels in fed and fasted long-chain acyl-CoA dehydrogenase (LCAD) KO mice. These mice mimic patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (33). As LCAD catalyzes the initial step in mitochondrial fatty acid oxidation, LCAD KO mice have impaired fatty acid oxidation and decreased fasting tolerance (34). We subjected LCAD KO mice to 16 h of food withdrawal to determine whether blunted fatty acid oxidation would have an effect on protein acetylation levels. In wild-type (WT) mice we observed a robust increase in hepatic protein acetylation (Fig. 6B). Strikingly, in fasted LCAD KO mice we did not detect hepatic protein hyperacetylation in response to fasting (Fig. 6B). The fact that we still observe some lysine acetylation in the LCAD KO mouse can be explained by oxidation of acyl-CoAs that are not LCAD substrates (35). This partial fatty acid oxidation is enough to maintain basal acetylation levels but is not sufficient to induce hyperacetylation.

Given that most of acetylation takes place in mitochondria and that SIRT3 is the main mitochondrial deacetylase in the liver (36,37); our observations could also be explained by a decrease in the levels of SIRT3. Therefore, we analyzed SIRT3 expression by western blot (Fig. 6C). SIRT3 expression increased slightly upon fasting in WT livers as has been reported previously (38). Thus the increase in acetylation upon fasting cannot be explained by a decrease in SIRT3 expression. Also, SIRT3 levels in fasted LCAD KO mice were not different from fasted WT mice, indicating that the observed lack of protein hyperacetylation in LCAD KO mice cannot be explained by an increase of SIRT3 expression (Fig. 6C).

Taken together, in mice that have fatty acid oxidation defects, fasting-induced protein hyperacetylation in liver is absent. This directly demonstrates that fatty acid oxidation is in fact essential for the hepatic hyperacetylation that takes place during fasting and thereby confirms our hypothesis in vivo.

DISCUSSION

In this study, we uncovered that acetyl-CoA dictates mitochondrial lysine acetylation during fasting in liver. We are the first to show that acetyl-CoA generated by fatty acid oxidation in liver is in fact necessary and sufficient to drive global protein hyperacetylation. In mice deficient in fatty acid oxidation, protein hyperacetylation was absent upon food withdrawal. Furthermore, we were able to trace acetyl units from radiolabeled palmitate to acetylated protein in fatty acid oxidation rich metabolic conditions, demonstrating that fatty acids are a source for acetylation during fasting. Overall, our data provide novel insights into mitochondrial lysine acetylation dynamics.

Given that acetyl-CoA is generated in large amounts in the mitochondria under metabolic conditions promoting fatty acid oxidation, global mitochondrial protein acetylation might be an inevitable by-product of metabolism. This idea is in line with other studies that have linked hepatic protein hyperacetylation to metabolic conditions accompanied by high fatty acid turnover in the liver. CR in mice is associated with higher fatty acid oxidation rates in the liver and results in increased acetylation of proteins (6,15). In addition, a high-fat diet and administration of ethanol in mice are associated with high lipid influx and steatosis in the liver. Both conditions also present with hyperacetylation of hepatic proteins (16,17).

The idea that hyperacetylation is driven by acetyl-CoA produced by fatty acid oxidation is also supported by the cellular compartmentalization of acetylation. In human liver we demonstrated that acetylation is most prevalent in both mitochondria and peroxisomes which are the two sole organelles that are able to catabolize fatty acids. Many other studies have established that acetylation is especially abundant in mitochondria (1,7), but we are the first to show that lysine acetylation is also abundant in the peroxisomes. It would be interesting to test whether proteins with lysine deacetylase activity (HDACs and sirtuins) reside in the peroxisomes and would impact peroxisomal physiology.

SIRT3 protein levels were shown previously to be upregulated during fasting and other conditions characterized by elevated fatty acid oxidation (6,15–17). If SIRT3 would be a general regulator of protein hyperacetylation, upregulation of SIRT3 would be expected to drive decreased acetylation. Instead, a more sophisticated mechanism is likely to be in place. The parallel upregulation of SIRT3 with hyperacetylation in the mitochondria can point to a mechanism in which SIRT3 would deacetylate specific lysines that were acetylated due to the high levels of acetyl-CoA in mitochondria. Indeed, numerous studies have shown that acetylation of specific lysines on mitochondrial enzymes reduces their activity, which can be restored through deacetylation by SIRT3 (8,10,38,39). Mice lacking SIRT3 do not exhibit apparent metabolic abnormalities under normal physiological metabolic conditions, however they are more sensitive to metabolic stresses such as long-term high-fat diet and non-physiological fasting (8,38). Furthermore, a number of studies have linked SIRT3 to regulation of ROS levels (11,40). Therefore, SIRT3 appears to protect the cell from stress imposed by changes in metabolic activity, in which case its absence could lead to negative physiological effects in long-term. Our observations that hyperacetylation is directly linked to increased fatty acid oxidation flux, highlight the importance of SIRT3 in overcoming aberrant protein acetylation events in mitochondria during metabolic stress.

The mechanism that underlies mitochondrial lysine acetylation remains to be resolved. Since most cytosolic and nuclear acetyltransferases operate at a Km that is comparable to the physiological acetyl-CoA concentration (41), increasing concentrations of acetyl-CoA could drive the acetyltransferase reaction in the forward direction. However, since the mitochondrial physiological conditions (relatively higher pH than other cellular compartments) makes a non-enzymatic reaction between acetyl-CoA and lysine more favorable, as can be observed in vitro (26), it is also possible that non-enzymatic lysine acylation is the dominant mechanism in mitochondria. Our study highlights the importance of acetyl-CoA dynamics in driving mitochondrial lysine acetylation, and provides detailed experimental evidence for a regulating link between metabolic pathways and lysine acetylation.

In summary, we provide a mechanism for mitochondrial acetylation dynamics and show that acetyl-CoA produced by fatty acid oxidation is driving protein acetylation. The implications of our proposed mechanism are that chronic metabolic imbalances characterized by enhanced fatty acid oxidation may well lead to protein hyperacetylation which in turn impacts on protein function and organismal physiology. Pharmaceutical boosting of SIRT3 activity would be beneficial to correct undesirable protein hyperacetylation under pathological conditions such as the metabolic syndrome.

MATERIALS AND METHODS

Animal studies

All animal experiments were carried out according to local, national and European Union ethical guidelines. LCAD−/− mice on a pure C57BL/6 background were maintained on a commercially available standard chow diet (CRM (E), Special Diets Services, Someren). Samples of fed and overnight fasted WT and LCAD−/− animals were obtained from a study performed by Houten et al. (42). For the fasting time course study, 8-weeks-old C57BL/6 mice were obtained from Harlan and were subjected at the age of 12 weeks to food withdrawal starting at 4 p.m. for 4 and 16 h. All mice were housed on a 12:12 light:dark cycle at 21°C (±2°C). Liver samples from PPARα WT and KO mice treated with WY14643 were obtained from a study performed by van Vlies et al (43). For this, PPARα WT or KO mice were fed ad libitum for 2 weeks with a diet containing 0.1% WY14643 or a control diet. For 3-hydroxybutyrate analysis, ethylenediaminetetraacetic acid (EDTA) blood (75 µl) was directly mixed with 75 µl 1 m perchloric acid, stored on ice for at least 10 min and frozen at −20°C until analysis of 3-hydroxybutyrate levels by LC-MS/MS. Blood glucose levels were analyzed in blood from saphenous vein using a glucometer.

SDS-PAGE, western blotting, immunoprecipitation

Tissue protein lysates were prepared using ultra-turrax homogenization in 50 mm Tris (pH 7.4), 0.5 mm EDTA, 0.1% Triton X-100 containing protease inhibitors (Roche) and deacetylases inhibitors (1 µm trichostatin A and 10 mm nicotinamide), followed by sonication and clearance of the lysate by centrifugation at 12 000g for 10 min at 4°C. Lysates were run on NuPAGE 4–12% gradient gels (Invitrogen), proteins transferred to nitrocellulose and immunodetected using antibodies specific for acetyl lysine (monoclonal #9681 and polyclonal #9441 Cell Signaling), actin (#14128 Abcam), GDH (#100–4158 Rockland), catalase [in house generated (44)], crotonase [in house generated (45)], electron-transferring-flavoprotein dehydrogenase (#110316 Mitosciences), endoplasmic reticulum protein 72 (#5033 Cell Signaling), LAMP1 (#9091 Cell Signaling), SIRT3 (#5490 Cell Signaling) and VDAC (#14734 Abcam). Odyssey IR dye secondary antibodies (Li-COR) were used for detection.

Acetyl lysine analysis in human fibroblasts

Human dermal fibroblasts were routinely cultured in DMEM supplemented with 10% (v/v) fetal calf serum, 2 mm glutamine and 1% (v/v/v) pen/strep/fungizone. For acetyl lysine analysis we incubated cells either in serum-free Eagle's minimal essential medium (MEM) supplemented with 400 µml-carnitine and 120 µm palmitate for 96 h [a metabolic condition characterized by high fatty acid turnover (29)] or in DMEM. After exposure, the cell pellet was resuspended in 50 mm NH4CO3 buffer containing deacetylase inhibitors (1 µm Trichostatin A and 10 mm nicotinamide) followed by sonication at 40 J/Ws. To digest the protein into amino acids, samples were incubated with pronase (53702, Calbiochem), at a protein to pronase ratio of 10:1, in 50 mm NH4CO3 for 4 h at 37°C. The reaction was stopped with 5 volumes of acetonitrile, 10 µl 2.5 mm D4-labeled l-lysine internal standard (DLM-2640, Cambridge Isotopes Laboratories) and 10 µl 10 µm D8-labelled acetyl lysine internal standard (D-6690, CDN Isotopes). The samples were briefly vortexed and centrifuged at 14 000 rpm, 4°C 10 for 10 min followed by solvent evaporation at 40°C under a gentle stream of nitrogen. Samples were then taken up in 0.01% heptafluorobutyric acid and analyzed with LC–MS/MS.

Acetyl lysine measurement using LC–MS/MS

Ten microliters of the sample extract was injected onto a BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters Corp. Milford MA) using a UPLC system consisting of an Acquity solvent manager with degasser and an Acquity Sample Manager with column oven (Waters Corp.). The system was controlled by MassLynx 4.1 software. The flow rate was set to 500 µl/min. Elution solvent A consisted of 0.1% heptafluorobutyric acid and solvent B was 80% acetonitrile. The chromatographic conditions were as follows: 0–2 min 100% A, 2–5 min to 50% B, 5–6 min to 100% B, at 6.1 min back to 100% A and equilibration time with 100% A was 3 min. Separation was performed at 50°C. The Quattro Premier XE triple-quadrupole mass spectrometer (Waters Corp.) was used in the positive electrospray ionization (ESI) mode. Nitrogen was used as nebulizing gas and argon was used as collision gas at a pressure of 2.5e−3 mbar. The capillary voltage was 3.0 kV, the source temperature was 120°C and desolvation temperature was 300°C. Cone gas flow was 50 l/h and desolvation gas flow was 900 l/h. All components were measured by using multiple reaction monitoring (MRM) in the positive ionization mode, using the transitions: m/z 147.0 > 84.1 for lysine, 151.0 > 88.1 for lysine-2H4 (internal standard), 189.2 > 84.1 for N-acetyl lysine and 197.2 > 91.1 for N-acetyl lysine-2H8 (internal standard) with optimal collision energy of 20 eV for lysine and 30 eV for N-acetyl lysine.

Human liver organellar fractionation

Liver was homogenized in 5 mm MOPS, 250 mm sucrose, 2 mm EDTA, 0.1% ethanol at pH 7.4 and centrifuged at 600g for 10 min at 4°C to obtain a PNS (46). The PNS was centrifuged at 3000g for 10 min at 4°C and the obtained supernatant was centrifuged again at 30 000g for 15 min at 4°C to produce an organelle-enriched fraction, which was subsequently resuspended in buffer supplemented with 5% Nycodenz. Following Nycodenz equilibrium gradient centrifugation, fractions were collected and equal amounts of protein were loaded on SDS-PAGE gels.

Radioactive tracing of acetylation from 14C-palmitate

Exposure conditions

Fao hepatoma cells were maintained on MEM supplemented with 2 mm glutamine, 1% non-essential amino acids, 2% Pen/Strep and 10% FBS. For metabolic exposure studies, FBS was dialyzed to deplete the medium of carnitine and glucose. For glucose-rich culture conditions 5.6 mm glucose was added to the medium, whereas for fatty acid culture conditions 5.6 mm galactose, 0.1% (w/v) bovine serum albumin (BSA), 100 µm palmitate and 50 µm carnitine were added to the medium. For overnight incubation of cells with 14C-labeled palmitate, three T-75 flasks with 80% confluent Fao cells were used per condition. Per flask 10 µCi of 14C-[1]-palmitate (ARC) was used. CO2 produced overnight was trapped to quantify the amount of label, which was then used as a measure of palmitate oxidation. The incubation medium was removed and the cells were washed twice with 4 ml BSA (fatty acid free)/phosphate buffered saline (2 g/l) to ensure the removal of remaining palmitate and twice with 4 ml PBS. The cells from each flask were scraped into 500 µl fractionation buffer [250 mm sucrose, 20 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm ethylene glycol tetraacetic acid (EGTA), 10 mm nicotinamide, 1 mm Trichostatin A and protease inhibitors (Roche)] and cell suspensions from the same condition were pooled.

Subcellular fractionation

Cytosolic and mitochondrial fractions were obtained by differential centrifugation. 1.5 ml cell suspension in fractionation buffer [250 mm sucrose, 20 mm Hepes, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 10 mm nicotinamide, 1 µm Trichostatin A and protease inhibitors (Roche)] was passed 10 times through 25G needle to disrupt the cells. The cell lysate was then centrifuged for 5 min at 700g at 4°C to obtain a PNS. PNS was centrifuged at 10 000g for 5 min at 4°C to obtain enriched cytosolic fraction in the supernatant and enriched mitochondrial fraction from the pellet. The pellet containing mitochondria was washed by resuspending it in fractionation buffer and letting it pass 10 times through the needle. Afterwards it was collected by centrifugation for 10 min at 10 000g at 4°C. To verify purity of cytosolic and mitochondrial fractions, LDH (cytosolic) and GDH (mitochondrial) activities were analyzed as described in in Bergmeyer (47). In short, LDH activity was measured in 50 mm KPi pH 7.4, 0.3 mm nicotinamide adenine dinucleotide (NADH) and 0.1% Triton. Pyruvate was used as the starting reagent. GDH activity was measured in 50 mm triethanolamide/HCl pH 8.0, 2.5 mm EDTA, 100 mm NH4Cl, 1 mm adenine dinucleotide phosphate, 0.3 mm NADH and 0.1% Triton. α-ketoglutarate was used as the starting reagent. Both activity assays were performed on Tecan Freedom Evo platform. Protein concentration was determined in cytosolic and mitochondrial fractions using BCA assay.

Immunoprecipitation of 14C-acetylated proteins

Cytosolic and mitochondrial fractions were diluted in immunoprecipitation (IP) buffer [50 mm Tris/HCl pH 7.4, 1% Triton, 150 mm NaCl, 0.5 mm EDTA, 10 mm NAM, 1 µm TSA and protease inhibitors (Roche)] to obtain 500 µg protein input for each IP. Samples were incubated 2 h with anti-acetylated lysine antibody (#9681 Cell Signaling) at 4°C followed by overnight incubation with magnetic protein A/G beads (Millipore). Supernatants were discarded and beads were washed four times with 1 ml of IP buffer. The acetylated proteins were eluted by boiling the beads in NuPage sample buffer and samples were loaded on SDS-PAGE 4–12% NuPage gradient gels (Invitrogen). After transferring the samples to nitrocellulose, the membrane was placed in a phosphorimager to capture the radioactive signal after 3–4 days exposure time.

Palmitate oxidation analysis

Palmitate oxidation was assessed by quantifying the formation of 3H2O from [9,10-3H(N)]-palmitate as described by (48). In short, the cells were plated out in a 48-well plate 18 h prior to the assay, with each condition performed in quadruplicate. The overnight medium was removed right before starting the assay. During the assay the cells were incubated in the same medium composition as overnight and 100 µm palmitate and a tracer amount of tritiated palmitate for 2 h at 37°C. Oxidation rate was expressed as nanomoles of fatty acid oxidized per hour per milligram of cell protein.

Acetylation of GDH using acetic anhydride

Glutamate dehydrogenase (bovine G7882, Sigma Aldrich) was incubated at 5 mg/ml with 0–5 mm acetic anhydride at room temperature in 50 mm ammonium bicarbonate (ABC) pH 8.3 for 15 min. During reactions, pH was monitored and adjusted to pH 8.3 with 1 m ABC. Reactions were stopped by adding NuPage sample buffer and boiling.

Statistics

Data are expressed as means ± SEM. Intergroup comparisons were performed by one-way ANOVA with Bonferroni post-tests or by Student's t-test when comparing two conditions. P < 0.05 was considered significant.

FUNDING

This work was supported by grants from The Netherlands Organization of Scientific Research (NWO), VIDI grant (016.086.336) to S.M.H. and NWO VENI grant (916.10.065) to V.C.J.d.B. and from the Academic Medical Center, Amsterdam, The Netherlands.

ACKNOWLEDGEMENTS

We thank Lodewijk IJlst, Simone Denis and Riekelt Houtkooper for helpful comments and technical support.

Conflict of interest. The authors declare no conflicts of interest.

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