A high-fat diet increases hepatic mitochondrial turnover through restricted acetylation in a NAFLD mouse model

Abstract

Lysine acetylation of proteins has emerged as a key posttranslational modification (PTM) that regulates mitochondrial metabolism. Acetylation may regulate energy metabolism by inhibiting and affecting the stability of metabolic enzymes and oxidative phosphorylation (OxPhos) subunits. Although protein turnover can be easily measured, due to the low abundance of modified proteins, it has been difficult to evaluate the effect of acetylation on the stability of proteins in vivo. We applied ²H₂O-metabolic labeling coupled with immunoaffinity and high-resolution mass spectrometry method to measure the stability of acetylated proteins in mouse liver based on their turnover rates. As a proof-of-concept, we assessed the consequence of high-fat diet (HFD)-induced altered acetylation in protein turnover in LDL receptor-deficient (LDLR^-/-) mice susceptible to diet-induced nonalcoholic fatty liver disease (NAFLD). HFD feeding for 12 wk led to steatosis, the early stage of NAFLD. A significant reduction in acetylation of hepatic proteins was observed in NAFLD mice, based on immunoblot analysis and label-free quantification with mass spectrometry. Compared with control mice on a normal diet, NAFLD mice had overall increased turnover rates of hepatic proteins, including mitochondrial metabolic enzymes (0.159 ± 0.079 vs. 0.132 ± 0.068 day^-1), suggesting their reduced stability. Also, acetylated proteins had slower turnover rates (increased stability) than native proteins in both groups (0.096 ± 0.056 vs. 0.170 ± 0.059 day^-1 in control, and 0.111 ± 0.050 vs. 0.208 ± 0.074 day^-1 in NAFLD). Furthermore, association analysis revealed a relationship between the HFD-induced decrease in acetylation and increased turnover rates for hepatic proteins in NAFLD mice. These changes were associated with increased expressions of the hepatic mitochondrial transcriptional factor (TFAM) and complex II subunit without any changes to other OxPhos proteins, suggesting that enhanced mitochondrial biogenesis prevented restricted acetylation-mediated depletion of mitochondrial proteins. We conclude that decreased acetylation of mitochondrial proteins may contribute to adaptive improved hepatic mitochondrial function in the early stages of NAFLD.NEW & NOTEWORTHY This is the first method to quantify acetylome dynamics in vivo. This method revealed acetylation-mediated altered hepatic mitochondrial protein turnover in response to a high-fat diet in a mouse model of NAFLD.

Keywords: NAFLD; acetylome dynamics; metabolic labeling; mitochondria; turnover.

Graphical abstract

Figure 1.

The study design and experimental scheme for the acetylome dynamics study. Eight-week-old male mice (n = 6 mice/group) were fed either a normal chow diet (control group) or a high-fat diet (HFD, NAFLD group) for 12 wk. At the ninth week of the diet experiment, the ²H₂O-based turnover study was initiated by a bolus injection of pure ²H₂O saline (20 μL/g of body wt) followed by 6% ²H₂O in the drinking water. Animals were euthanized at specific time points up to 21 days. The liver was removed, and proteins were isolated and digested with trypsin (see “Experimental Procedures”). After immuno-enrichment of acetylated peptides, native and acetylated peptides were subjected to LC-MS/MS analysis. The kinetics of acetylated and native peptides were analyzed based on ²H-incorporation into tryptic peptides. LC-MS/MS, liquid chromatography-tandem mass spectrometry; NAFLD, nonalcoholic fatty liver disease.

Figure 2.

Twelve weeks of HFD consumption results in impaired glucose utilization and induces steatosis in LDLR^−/− mice. A: glucose tolerance test. HFD results in impaired glucose utilization. Data represent means ± SD (n = 4/group). *P value < 0.05. B and C: representative hematoxylin and eosin (H&E)-stained liver sections (×20 magnification). Livers from NAFLD mice show the presence and distribution of accumulated fats (C) which was not visible in the livers of control mice (B). HFD, high-fat diet; NAFLD, nonalcoholic fatty liver disease.

Figure 3.

HFD-induced NAFLD suppresses acetylation of hepatic proteins in male mice. A: lysine-acetylated proteins extracted from control and NAFLD liver homogenate. B: lysine-acetylated proteins in the cytosolic fractions from control and NAFLD mouse livers. C: lysine-acetylated proteins in the mitochondria from control and NAFLD mouse livers. Gel images were quantified relative to the respective loading controls by densitometry using ImageJ 1.41 software. Normalized intensities for all replicates (6 male mice/group) are shown in boxplots. The P values are based on two-sample t tests to compare mean protein levels between the control and NAFLD groups. *P value < 0.05. HFD, high-fat diet; NAFLD, nonalcoholic fatty liver disease.

Figure 4.

Proteomics analysis of acetylation. A and B: Venn diagram of acetylated proteins and peptides identified in control and NAFLD groups. A total of 993 and 862 acetylated peptides representing 398 and 366 proteins were identified in control and NAFLD groups, respectively. Many of these peptides with the same acetylation sites (630 total) representing 288 proteins were common in both groups. C: label-free quantification of acetylated peptides. The volcano plot shows differential expression of acetylated peptides between the NAFLD and control groups, in terms of their log₂ fold changes (x-axis) and −log₁₀ transformed adjusted P values (y-axis). The horizontal dashed line indicates the 0.05 threshold of the FDR-adjusted P value; the vertical dashed lines indicate twofold increase or decrease. Significantly upregulated and downregulated acetylated sites with an adjusted P value <0.05 are shown in red and black colors, respectively. Significant sites with at least twofold increase or decrease are labeled with their gene names and site locations. FDR, false discovery rate; NAFLD, nonalcoholic fatty liver disease.

Figure 5.

Acetylation increases the half-life of ATP/ADP translocase 2 (ADT2), a mitochondrial protein, in control mice. A: turnover rates of native and acetylated ADT2 forms. ²H-labeling of native and acetylated ADT2 peptides enables us to determine the newly made fraction at each time point. B: estimated turnover rates of native and acetylated ADT2 peptides and their standard errors (shown in error bar) based on data from A. The P value (< 0.05, marked with an asterisk) is based on the F test to evaluate the change in turnover rate between the acetylated and native peptides.

Figure 6.

NAFLD- and acetylation-related changes in turnover rates of proteins in mouse liver. A: volcano plot summarizing NAFLD-related changes in turnover rates for native proteins (gray) and acetylated sites (red). The x-axis represents the fold change in turnover rate of NAFLD with respect to control (on log₂ scale), and the y-axis represents the FDR-adjusted P value (on -log₁₀ scale) for the change. The turnover rates were estimated based on all the quantified peptides corresponding to the same native protein or acetylated site. The horizontal dashed line indicates 0.05 FDR-adjusted P value, the threshold for statistical significance; the vertical dashed lines indicate twofold increase or decrease in turnover rate. Seven acetylated sites with a significant increase by at least twofold are labeled with their gene names and site locations. B: volcano plots summarizing acetylation-related changes in turnover rates, shown separately for the control and NAFLD groups. The acetylation-related change was determined by comparing the turnover rates of paired acetylated and native peptides. Each dot represents an acetylated lysine site. Acetylated sites that change significantly in turnover rate (adjusted P value <0.05, at least two-fold increase or decrease) are shown in black color. There are 20 sites on 13 proteins with a significantly decreased turnover in both control and NAFLD groups, and they are labeled with their gene names and site locations. C: protein-level summary of turnover rate. Each dot represents a native (gray) or acetylated protein (red). The turnover rate of an acetylated protein is calculated by averaging over the turnover rates of the associated acetylated peptides. FDR, false discovery rate; NAFLD, nonalcoholic fatty liver disease.

Figure 7.

Association between NAFLD-related changes in acetylation level and turnover rate. Each dot represents an acetylated site. The x-axis represents the fold change in acetylation of NAFLD with respect to control (on log₂ scale), and the y-axis represents the NAFLD-related fold change in turnover rate (on log₂ scale) of a peptide acetylated at the specific lysine site, as shown in the x-axes of Figs. 4C and , respectively. There are 14 sites on 9 proteins with a significant change in both acetylation level and turnover rate (adjusted P value <0.05), among which 11 and 3 had a significant decrease and increase, respectively, in acetylation. These sites are shown in black or red color and labeled with their gene names and site locations. NAFLD, nonalcoholic fatty liver disease.

Figure 8.

g:Profiler functional enrichment analysis by querying the proteins with statistically significant changes in turnover rates. A: significantly enriched terms from Reactome (adjusted P value <0.05) with size <500 shown in bars. The length of each bar represents the statistical significance (on the −log₁₀ scale) of the associated term. The number of proteins involved in each term and the term size are shown on the right of each bar. B: protein-level summary of turnover rate to summarize the effect of acetylation and NAFLD, shown separately for four significantly enriched Reactome terms: the citric acid (TCA) cycle and respiratory electron transport (TCA cycle and ETC), mitochondrial biogenesis, metabolism of amino acids and derivatives (amino acid metabolism), and formation of ATP by chemiosmotic coupling (ATP synthesis). ETC, electron transport chain; NAFLD, nonalcoholic fatty liver disease; TCA, tricarboxylic acid.

Figure 9.

Effect of HFD on expression of mitochondrial proteins. A: immunoblot analysis of respiratory chain proteins in isolated mitochondria. Proteins from isolated hepatic mitochondria (20 µg) were fractionated on SDS-PAGE gel (4–12%) and transferred to PVDF membrane. Western blot analysis was performed using antibodies for NDUFB8 (CI subunit), SDHB (CII subunit), UQCR2 (CIII subunit), and anti-ATPA (CV subunit). B: boxplots of the expression levels of mitochondrial proteins after normalization relative to ATPA. C: immunoblots of TFAM expression in the liver from control and NAFLD mice. D: boxplots of the relative levels of TFAM, mitochondrial transcription factor. Boxplots in B and D are overlaid with normalized intensities for all replicates (6 male mice/group). The P values are based on two-sample t tests to compare mean protein levels between the control and NAFLD groups. *P value < 0.05. ATPA, ATP synthase subunit α; HFD, high-fed diet; NAFLD, NAFLD, nonalcoholic fatty liver disease; SDHB, succinate dehydrogenase B; TFAM, mitochondrial transcriptional factor.