Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity

Cell culture and adipogenic differentiation

The preadipocyte cell line, 3T3-L1, which is derived from mouse embryonic fibroblasts, was purchased from ATCC. The cells were cultured in growth medium (high glucose DMEM containing a 1% antibiotic-antimycotic solution and 10% bovine calf serum; Gibco-Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere with 5% CO₂. GP2-293 packaging cells were grown in DMEM containing a 1% antibiotic-antimycotic solution and 10% FBS. 3T3-L1 cells were induced to differentiate into mature adipocytes as described in our previous reports (18–21). Confluent 3T3-L1 cells were incubated in differentiation medium composed of DMEM, 10% FBS, and MDI (a differentiation cocktail of 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg/ml insulin [Sigma, US]). After 48 h, the medium was changed to a maintenance medium composed of DMEM, 10% FBS, and 10 μg/ml insulin. The medium was replenished every other day.

Oil-Red-O staining

Cultured cells were washed twice with PBS and fixed with 10% formalin for 30 min at room temperature. The cells were then washed with distilled water and stained for 30 min at room temperature with 0.3% filtered Oil-Red-O solution in 60% isopropanol (Sigma, St. Louis, MO). The stained cells were washed with distilled water, and micrographs were obtained. To extract the incorporated Oil-Red-O dye, absolute isopropanol was added to the stained cell-culture dish, and the dish was shaken at room temperature for 30 min. Triplicate samples were read at 510 nm using a GeneQuant 1300 spectrophotometer (GE HealthCare, Uppsala, Sweden) (20, 21).

Sample preparation and 2D-electrophoresis

The proteins were extracted from 3T3-L1 cells using a lysis buffer (30 mM Tris [pH 8.5], 7 M urea, 2 M thiourea, and 4% 3-[(3-cholamidopropyl)-dimethylammonio propanesulfonate [CHAPS]). Protein samples to be separated on the same gel were pooled, and 2 volumes of cold acetone were added. After centrifugation, the precipitate was rehydrated using a rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% ampholyte, and 20 mM DTT), and isoelectric focusing was conducted using a Multiphor II apparatus (GE HealthCare). After focusing, each strip (a 7 cm immobilized pH nonlinear strip [pH 3–11]) was equilibrated twice for 15 min in 2.5 ml of equilibrium buffer. The equilibrium buffer contains 50 mM Tris (pH 8.8), 6 M urea, and 30% glycerol. During the second equilibrium step, 260 mM iodoacetamide was added to the equilibrium buffer. The IPG strips were then loaded and separated on a 10% acrylamide SDS-PAGE gel using a Mini-Protean system (Bio-Rad, Hercules, CA).

Two-dimensional gel electrophoresis Western blot analysis

Sixty micrograms of total protein was used for the detection of lysine acetylated proteins during adipogenic differentiation. The samples were separated in the first and second dimension following previously described protocols (22, 23) and transferred to an Immunoblot PVDF membrane (Millipore). Membranes were blocked with 5% skim milk for 1 h and probed with a rabbit anti-acetyl-lysine antibody (ICP0380 Immunechem) diluted 1:2,000 in TBS with 5% skim milk for 4 h at 4°C as previously described (21).

Protein digestion, peptide enrichment, and MS analysis

The separated proteins in SDS-PAGE gels were visualized by Coomassie brilliant blue G-250 staining, and spots of interest were detected. Gel pieces were washed twice with 150 μl of 100 mM ammonium bicarbonate (pH 8.2) and 70% v/v acetonitrile and dried at 37°C for 20 min. Trypsin in 50 mM ammonium bicarbonate (20 μg/μl) was added to each gel piece, and gel pieces were incubated at 37°C for 2 h. Peptides were then extracted using a mixture containing 20 μl of 0.1% v/v trifluoroacetic acid and 70% acetonitrile. The peptides were purified using ZipTips^TM (Millipore) according to the manufacturer's instructions before LC-MS/MS spectrometric analysis. The peptides were analyzed using a Synapt High Definition mass spectrometer (Waters, Manchester, UK) as previously described (24, 25).

Construction of retroviral vectors and transduction

To construct 3T3-L1 cells that stably express a FLAG-tagged wild-type or mutant MDH1 protein, a retroviral infection system was used. For the expression of MDH1, DNA encoding the FLAG-tagged MDH1 was inserted into 3T3-L1 cells using the pRetroX-IRES-ZsGreen1 vector (Clontech). For virus production, GP2-293 cell lines were transfected using Lipofectamine 2000 (Gibco-Invitrogen). The details of the transfection and transduction methods have been described in our previous reports (18–21, 26). Infected cells were selected using a FACSAria cell sorter (BD Biosciences, San Jose, CA) and further maintained in growth medium. The ectopic expression of MDH1 was confirmed by Western blot analysis.

Introduction of mutations at putative acetylation sites

FLAG-tagged MDH1 was mutated using the EZchange^TM Site-Directed Mutagenesis kit (Enzynomics, KR). The putative acetylation sites in MDH1 were mutated to arginine or glutamine. Arginine is charged and abolishes acetylation, whereas glutamine is uncharged and may mimic the acetylated lysine. The sites mutated in this study were based on those reported by Zhao et al. (K118, K121, and K298) (5). We introduced the mutation into each site individually or at all three sites of MDH1.

Construction and purification of mutant MDH1 proteins in Escherichia coli

Sequences from the wild-type and mutant MDH1 gene were amplified using the forward primer 5′- gggaattccatatgatgtctgaaccaatcagagtc-3′ containing an NdeI site (in bold) and the reverse primer 5′-ccgctcgagttaggcagaggaaagaaattcaaa-3′ containing a XhoI site (in bold) and inserted into the corresponding sites of the pET28a plasmid (Novagen, Madison, WI). Escherichia coli Rosetta DE3 cells (Novagen) carrying the respective expression plasmids were grown at 37°C in LB medium until the optical density at A₆₀₀ reached 0.6–0.8. The expression of MDH1 and MDH1 mutants was induced by adding 1 mM IPTG at 18°C for 18 h. The cells were harvested, washed with lysis buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 5% glycerol, and 1 mM β-mercaptoethanol), and lysed by ultrasonication. After centrifugation (29,820 g for 30 min), the supernatants were incubated with a cobalt affinity resin (TALON^®, Clontech) on a rocker for 1 h at 4°C and washed with lysis buffer supplemented with 10 mM imidazole. The proteins were eluted from the metal affinity resin using a lysis buffer supplemented with 100 mM imidazole. The proteins were concentrated to 5 mg/ml and stored at −80°C.

Immunoblotting and immunoprecipitation

The cells were washed three times with ice-cold PBS and were harvested in ice-cold 7 M urea lysis buffer containing a protease inhibitor cocktail (Roche). The protein concentrations were measured using a Bradford assay (Bio-Rad). Immunoblotting and immunoprecipitation were performed as described previously. The anti-MDH1 antibody was obtained from Santa Cruz, and the acetyl-lysine antibody was purchased from ImmuneChem Pharmaceuticals Inc. Anti-FLAG and anti-α-tubulin antibodies were purchased from Sigma. Anti-enolase1 and anti-histone H3 antibodies were obtained from Cell Signaling (Beverley, MA). The secondary antibodies were purchased from Abcam. The membranes were visualized using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL).

For immunoprecipitation, anti-acetyl-lysine antibody agarose beads (ImmuneChem Pharmaceuticals Inc.) were added to 200 μg of the lysates. The mixture was incubated overnight at 4°C. The immunoprecipitates were recovered by centrifugation at 2,500 g, washed five times with NP-40 lysis buffer, and analyzed as described above.

Activity assay of MDH1

Cell pellets were lysed with NP-40 buffer supplemented with a protease inhibitor (Roche, Indianapolis, IN) and a KDAC inhibitor (10 µM trichostatin A, 10 mM nicotinamide, and 50 mM butyric acid; Sigma, US) (5). For immunoprecipitation, extracted proteins were incubated with anti-FLAG M2 agarose beads (Sigma) at 4°C overnight. The immunoprecipitated beads were washed 10 times with NP-40 buffer, and then the FLAG fusion proteins were eluted using a 3×FLAG peptide (Sigma). The concentration of the eluted proteins was measured using a Bradford assay and Coomassie blue G-250 staining. Purified human MDH1 that was overexpressed in 3T3-L1 cells was incubated with 0.2 mM oxaloacetate and 1 mM NADH in PBS (pH 7.5). The activity of MDH1 was determined by measuring the decrease in the fluorescence of NADH (Ex. 350 nm; Em. 470 nm). The reaction was monitored 15 times at 1-min intervals on a Victor^TM X3 Multilabel plate reader (PerkinElmer, Waltham, MA).

Analysis of intracellular NADPH levels

Commercial NADP/NADPH assay kits (Abcam, Cambridge, UK) were used to detect NADPH and total NADP. The cells were washed three times with ice-cold PBS and placed in NADP/NADPH extraction buffer. The washed cells were extracted by two freeze (20 min on dry-ice)/thaw (10 min at room temperature) cycles, and then the cell lysates were dissolved in buffer using gentle sonication. The concentration of the eluted proteins was measured using a Bradford assay (Bio-Rad). To calculate the NADP/NADPH ratio, total NADP (NADPt) and NADPH-only were prepared, and an NADPH standard was made using a serial dilution of NADPH. The NADPH reaction was monitored on a microplate reader (Bio-Rad) for 1 to 4 h at 450 nm.

Analysis of acetylome changes during adipogenesis

Many groups have reported that the adipogenic differentiation process could be differentially regulated depending on the type of HDAC inhibitors, suggesting that acetylation may be intimately associated with adipogenesis (9–11). As an initial step, 1D SDS-PAGE was conducted to determine whether acetylation patterns changed during adipogenesis (Fig. 1A). Whole cell proteins from 3T3-L1 cells derived from 0, 2, and 12 days after the induction of differentiation were used for analysis. It was confirmed that the level of aP2 was increased after adipogenic stimulation (Fig. 1B). The expression level of histone H3 was unchanged during differentiation, and it was thus used as a loading control. Our results clearly demonstrated that the acetylation pattern and protein expression were significantly altered during the differentiation of 3T3-L1 preadipocytes into adipocytes. Next, we performed two-dimensional gel electrophoresis (2-DE). The expression pattern of proteins with isoelectric points ranging between pH 3 and 11 was analyzed. The overall changes in the acetylation pattern (acetylome) were also investigated by Western blot analysis using an anti-acetyl-lysine antibody after 2-DE (Fig. 1C). As shown, the levels of lysine-acetylated proteins were generally increased after adipogenic differentiation. A total of 69 spots showing changes in acetylation during adipogenesis were detected by image analysis and visual confirmation; these spots were identified by LC-MS/MS analysis (Table 1). As expected, a variety of metabolic enzymes, including those involved in glycolysis, fatty acid metabolism, and the TCA cycle, were acetylated during adipogenesis.

To validate the results of the acetylome analysis, we assessed the expression and acetylation level of several of the identified proteins in 3T3-L1 cells using Western blot and immunoprecipitation analyses, respectively (Fig. 2A). MDH1, one of the identified candidates, showed an approximately 2-fold increase in expression during adipogenesis. The acetylation level of MDH1 was also dramatically enhanced (6-fold) during adipogenic differentiation (Fig. 2B). MDH2 also demonstrated this enhanced acetylation pattern, but enolase showed no changes in expression or acetylation level during adipogenesis. Generally, the Western blot and immunoprecipitation results correlated well with the 2-DE acetylome data (data not shown).

Effect of the ectopic expression of MDH1 on adipogenic differentiation in 3T3-L1 preadipocytes

To examine if the acetylation of MDH1 occurred during adipogenesis, we assessed the expression and acetylation levels of MDH1 in other differentiation processes, such as myogenesis and osteogenesis. There was no significant change in expression and acetylation levels during myogenesis and osteogenesis (data not shown). These results suggest that the acetylation of MDH1 occurred in adipogenic differentiation. Next, to clarify the role of MDH1 acetylation in adipogenesis, 3T3-L1 cells were infected with the FLAG-tagged, full-length human MDH1 protein using a retroviral infection system (IRES-GFP). A control vector was used as the negative control. Infected cells were isolated by a FACS sorter, and most of the cells were GFP positive by fluorescence microscopy (Fig. 3A). The overexpression of MDH1 proteins was confirmed by Western blot analysis using anti-FLAG and anti-MDH1 antibodies (Fig. 3B). Infected cells were induced to differentiate into mature adipocytes, and after 8 days, lipid accumulation was assessed by Oil-Red-O staining of control vector- and MDH1-expressing cells (Fig. 3C). The results clearly showed that the overexpression of MDH1 significantly increased lipid accumulation when compared with the control vector (Fig. 3C, D). Consistently, the level of aP2 in the MDH1-infected cells was higher than that in the control vector-expressing cells (Fig. 3E). In addition, the expression levels of adipogenic markers, such as C/EBPα and PPARγ, were also increased upon ectopic expression of MDH1.

Effect of the mutation(s) of putative acetylation sites in MDH1 on adipogenic differentiation

To examine the effect of MDH1 acetylation on adipogenesis, putative acetylation sites in MDH1 were mutated to arginine, which is charged but blocks acetylation, or to glutamine, which is uncharged and may mimic the acetylated lysine (2, 4, 5, 27). K118, K121, and K298 were chosen for mutation to arginine or glutamine. 3T3-L1 cells were then infected with these mutants of MDH1 using a retroviral system (IRES-GFP). The control vector was used as a negative control, and wild-type MDH1 was used as a positive control. Infected cells were isolated using a FACS sorter. Most of the infected cells were GFP positive by fluorescence microscopy (Fig. 4A). The overexpression of the mutated MDH1 proteins was confirmed by Western blot analysis using anti-FLAG and anti-MDH1 antibodies (Fig. 4B). Infected cells were cultured in differentiation medium for 8 days, and lipid accumulation was assessed by Oil-Red-O staining. Among the MDH1 mutants, the 3KR (in which the three putative acetylation lysine residues were replaced with arginine) and K118R mutants showed a significantly lower level of Oil-Red-O staining than the cells infected with wild-type MDH1 (Fig. 4C, D). These results clearly demonstrate that the mutation of the acetylation site(s) influences adipogenic differentiation, indicating that the acetylation of MDH1 is intimately involved in controlling adipogenic differentiation. We confirmed the continuous overexpression of MDH1 until the late stage of adipogenic differentiation. In addition, we found that differences in adipogenic differentiation correlated well with the expression level of aP2 (Fig. 4E, F). The acetylation levels of 3KR and K118R were considerably lower than those of wild-type MDH1 (Fig. 4G, H). Next, we measured the mRNA level of adipogenic markers, such as adiponectin, resistin, C/EBPα, and PPARγ, using real-time PCR. As expected, the expression levels of these adipogenic markers were significantly increased by introducing the MDH1. Additionally, 3T3-L1 cells carrying 3KR or K118R showed the reduced expression levels of adipogenic markers, compared with wild-type MDH1 (Fig. 4I). To rule out the possibility that the mutation of MDH1 affects its enzymatic activity, we expressed and purified the wild-type and mutant MDH1 proteins (3KR and K118R) using the E. coli expression system. Mutant MDH1s have similar activity to wild-type MDH1, indicating that the introduction of the mutation has no effect on enzymatic activity (data not shown).

Activity assay of MDH1

An immunoblot analysis showed that the acetylation level of MDH1 increased during adipogenesis (Fig. 5A). Generally, acetylation modulates enzymatic function by altering enzymatic stability, catalytic activity, or protein-protein interactions. To explore the direct effect of acetylation on MDH1 activity, we purified the ectopically expressed wild-type MDH1 during adipogenesis (day 0 and day 8). MDH1 activity was increased by more than approximately 50% 8 days after differentiation when compared with the activity at day 0 (Fig. 5B). We then investigated the activity of the ectopically expressed wild-type and mutant MDH1 proteins after immunopurification. MDH1-3KR and MDH1-K118R showed significantly reduced enzymatic activity when compared with the wild-type and KQ mutant (Fig. 5C, D). Furthermore, the acetylation level of MDH1-3KR and MDH1-K118R considerably decreased when compared with wild-type MDH1 (Fig. 5E). These results clearly indicate that the acetylation level of MDH1 is enhanced during adipogenesis and that the enhanced acetylation of MDH1 increases its enzymatic activity.

Analysis of intracellular levels of NADPH after the introduction of MDH1 or mutant MDH1

Intracellular NADPH is an important source of reducing power for fatty acid synthesis. As mentioned above, MDH1 participates in the malate/aspartate shuttle that contributes the supply of acetyl-CoA and NADPH for fatty acid synthesis in the cytoplasm. To elucidate the mechanism of action of acetylated MDH1 on adipogenic differentiation, the NADP/NADPH ratio was assessed in cells 8 days after differentiation. The NADP/NADPH ratio was considerably decreased when wild-type MDH1 was overexpressed (Fig. 5F, G). In contrast, MDH1-3KR and MDH1-K118R showed a roughly similar ratio of NADP/NADPH to the control vector (Fig. 5F, G). These results strongly indicate that there is a relationship between the NADP/NADPH ratio and the enzymatic activity of MDH1, which is regulated by acetylation during adipogenesis.