Identification of multiple proteins whose synthetic rates are enhanced by high amino acid levels in rat hepatocytes
Abdul Jaleel and
K. Sreekumaran Nair
Endocrinology Research Unit, Mayo Clinic, Rochester, Minnesota 55905
Submitted 8 September 2003
; accepted in final form 3 February 2004
 |
ABSTRACT
|
---|
Amino acids are key regulators of protein synthesis in liver. However, it remains to be determined whether amino acids stimulate synthesis of all or certain specific liver proteins. No techniques are currently available to simultaneously measure synthetic rates of several individual proteins. Here we report studies performed on rat hepatocyte primary cultures in which we used metabolic labeling with [14C]leucine, two-dimensional gel electrophoresis (2DGE), and tandem mass spectrometry to identify proteins that showed increased leucine incorporation when high amino acid levels were present in the media. Rat hepatocytes were isolated by in situ collagenase perfusion, cultured in serum-free medium containing insulin, and incubated for 2, 4, and 8 h in media of standard and high amino acid concentrations. SDS-PAGE and 2DGE were performed to separate proteins from cell lysates. Proteins that consistently showed increased synthesis on triplicate cultures, as detected by phosphorimaging of gels, were identified by tandom mass spectrometry. The combination of these approaches enabled the detection of 16 specific liver proteins whose synthetic rates were enhanced by increased amino acid concentration. These proteins are involved in specific functions such as translation intiation, protein folding and modification, oxidative phosphorylation, antioxidant defense, signal transduction, and transport, as well as cell motility and tissue integrity. No quantitative changes for any of these proteins were detected by gel staining, indicating that no detectable changes in protein concentration occurred. In contrast, measurable changes in synthetic rates occurred in 16 proteins. In conclusion, amino acids stimulate the synthesis of several liver proteins with important cellular functions.
protein synthesis; liver proteins; metabolic labeling; hepatocyte culture; proteomics
THE PRESENT STUDY WAS DESIGNED with two specific aims. Primarily, we proposed to develop a technique to measure synthetic rates of several individual proteins in primary liver cell culture. Second, we sought to identify individual liver proteins whose synthetic rates are enhanced by increasing the supply of amino acids. Both amino acids and insulin have anabolic roles. After a mixed meal, the liver is exposed to high levels of amino acids and insulin. The regulatory roles of amino acids and insulin on liver protein synthesis have been investigated extensively. Studies conducted in humans (31) and animals (33) demonstrate that amino acids play a pivotal role in postprandial protein anabolism. In healthy humans, elevating plasma amino acids by infusion while maintaining insulin level at basal concentration results in increased protein synthesis across the splanchnic bed (26). Furthermore, an increase in splanchnic protein synthesis was shown in type 1 diabetic patients while plasma amino acid levels were elevated during insulin deprivation (25). Together, these studies indicate that amino acids independently enhance synthesis of proteins in the splanchnic bed. The increase in protein synthesis in the splanchnic bed observed in type 1 diabetic patients during insulin deprivation does not occur in the gut mucosa (6), suggesting that it occurs in the liver. Studies performed in a swine model have demonstrated that the increase in the synthesis of protein occurring in liver is not in the structural proteins, suggesting that synthesis of secretary proteins in liver is mediated by amino acids (1). Experiments performed in perfused rat liver have demonstrated that deprivation of essential amino acids causes inhibition of protein synthesis (10). Studies conducted in cultures of isolated rat hepatocytes have shown that both translational and pretranslational mechanisms are involved in liver protein synthesis induced by amino acids (21). Thus the in vivo and in vitro studies clearly indicate the stimulatory role of amino acids on liver protein synthesis. Most of these studies are confined to the measurement of average rates of synthesis of total liver proteins. It remains to be determined whether syntheses of all liver proteins or only certain specific proteins are stimulated by amino acids. One previous study has shown that insulin deficiency enhances synthesis of specific proteins, such as fibrinogen, whereas that of albumin is inhibited, indicating the differential effect of insulin on liver protein synthesis (8). Currently, it is not known whether amino acids have any differential effect on synthesis of various liver proteins.
To demonstrate changes in protein concentrations during a short experimental period requires highly sensitive techniques to quantify the separated proteins. Techniques such as two-dimensional gel electrophoresis (2DGE) and mass spectrometry are used to profile proteins from biological samples. Comparative analyses of stained 2DGE images have been used extensively to identify proteins that are differentially expressed. However, this approach is not capable of identifying small changes in protein concentrations during physiological intervention such as changing concentrations of amino acids. There are techniques to quantify protein concentration more accurately by the addition of fluorescent labels (30) or by labeling with an isotope-coded affinity tag reagent (13) to protein samples before 2DGE, followed by quantification by mass spectrometry. In these approaches, protein labeling can be carried out only after protein extraction; as a result, investigations are limited to the comparison of tissue or cellular total protein content. Current techniques, therefore, are not sufficiently sensitive to detect small changes in the concentrations of many proteins occurring during a short intervention period or proteins whose concentrations may not change because of their rapid turnover rate. The concentration of a specific protein is determined by the balance between the rates of synthesis and breakdown (proteolysis). It is likely that many proteins with important biological functions may not show substantial changes in concentration, even though their synthetic rates are altered considerably. In the present study, we report profiling of specific liver proteins whose synthetic rates are stimulated by amino acids. We used an approach of metabolic labeling of liver proteins by radioisotope-labeled leucine in isolated rat hepatocytes, coupled with 2DGE and mass spectrometry, to test the hypothesis that increased amino acid concentration per se increases synthesis of specific liver proteins.
 |
EXPERIMENTAL PROCEDURES
|
---|
Rat hepatocyte culture.
The experimental animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Mayo Clinic, Rochester, MN. Hepatocytes were isolated from adult male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing
200 g, according to the two-step collagenase perfusion technique described by Berry and Barritt (4), with slight modification. Animals were anesthetized using an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The abdominal cavity was then completely exposed, and the liver was isolated from surrounding tissue and fascia. The portal vein was catheterized using PE-50 tubing (Becton Dickinson, Bedford, MA), and an incision into the inferior vena cava was made to facilitate continuous perfusion of the liver with a peristaltic pump (Millipore, Billerica, MA) at a rate of 30 ml/min. The initial perfusate used to flush the blood from the liver was a HEPES-based buffer (33 mM HEPES, 160.6 mM NaCl, 3.15 mM KCL, 0.7 mM Na2HPO4) that had been warmed to 37°C and supplemented with 100,000 U of pen-strep and 0.5 mM EDTA. This buffer was perfused through the liver until the perfusate ran clear (
10 min). The perfusion was then stopped and the perfusate changed to a HEPES buffer containing 1 mM CaCl2 and 0.025% collagenase (Sigma Chemical, St. Louis, MO). When the liver appeared to be dissociated (
10 min) and while the integrity of liver capsule was still maintained, the liver was removed, placed in 20 ml of hepatocyte wash medium (GIBCO, Carlsbad, CA), and then maintained at 4°C throughout the cell isolation and washes. After the final wash, the cells were resuspended in Hepato-STIM (Becton Dickinson) containing 5% fetal calf serum (FCS). Cell viability and integrity were assessed by the trypan blue dye exclusion method. Hepatocytes were plated in specially charged six-well plates coated with 150 µl of Matrigel matrix (Becton Dickinson) at a cell density of 0.5 x 106 cells/well in 2 ml of Hepato-STIM with 5% FCS. Cells were incubated at 37°C and 5% CO2 for 4 h to allow the hepatocytes to adhere, and then the medium was exchanged for fresh serum-free Hepato-STIM containing 2 mM glutamine. Medium was changed every 24 h after plating until the experimental treatment.
Isotope tracers.
Cultured hepatocytes were incubated for 2, 4, and 8 h in media containing standard (i.e., 0.5 mM leucine) and high (i.e., 2 mM leucine) concentrations of amino acids with l-[U-14C]leucine (Amersham Pharmacia Biotech UK, Buckinghamshire, UK) at a radioactive concentration of 0.5 µCi/0.5 mM cold leucine. The specific activity of l-[U-14C]leucine was 277 mCi/mM. For the amino acid addition, we used 50x RPMI 1640 amino acid solution (Sigma). The composition of amino acids in the control (C) medium as well as high amino acid (AA) medium is shown in Table 1. After each incubation period, cells were washed with phosphate-buffered saline (PBS), and MatriSperse (Becton Dickinson) was added to the plates at 4°C to dissolve the MatriGel. Cell samples were washed three times with ice-cold PBS, and the cell pellets were stored at 80°C until analysis.
SDS-PAGE and 2DGE.
The cells (pellets) were dissolved in lysis buffer containing 35 mM Tris base, 9 M urea, 4% CHAPS {[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate}, 65 mM DTT, 0.2% 310 pH ampholyte, and trace bromophenol blue. Protein concentration was estimated by the Bradford method. Protein (20 µg) from each sample was loaded on a 12% SDS-polyacrylamide gel and run under reducing conditions in a Hoefer gel apparatus (model SE 600, Amersham Bioscience) at 100 V until the dye front reached the bottom of the gel. The gels were stained with Coomassie blue (GelCode Blue Stain Reagent; Pierce, Rockford, IL) and dried on filter paper with a gel dryer.
For 2DGE,
20 µg of each cell lysate protein were dissolved in the same lysis buffer to a final volume of 185 µl. These samples were used to rehydrate 11-cm-long pH 310, immobilized pH gradient (IPG) strips (Bio-Rad Laboratories, Hercules, CA) in a rehydration tray overnight. The rehydrated IPG strips were subjected to isoelectric focusing (IEF) in a Protean IEF Cell (Bio-Rad) by use of a three-step protocol. The focusing was achieved with an initial step of 250 V for 15 min and continued with a maximum of 8,000 V increased linearly from 250 V over 5 h in the second step and a third step to continue at 8,000 V for 48,000 volt hours. The cell temperature was kept at 20°C with a maximum current of 50 µA/strip. The IEF-preformed IPG strips were equilibrated for the SDS-PAGE in a two-step equilibration using 4 ml of equilibration buffer per strip (6 M urea, 2% SDS, 0.375 M Tris·HCl, pH 8.8, and 20% glycerol), with 130 mM DTT in the first step and 135 mM iodoacetamide in the second step. The equilibration steps were done in an equilibration tray for 10 min each on a rotary shaker at room temperature. The second-dimension separation by subunit molecular weight was performed by vertical SDS-PAGE. For 11-cm-long IPG strips we used the Criterion electrophoresis system and 816% precast gels of size 9 x 13 cm (Bio-Rad). The medium-sized gels were chosen for second dimension by exposing the gels of C and AA of each incubation time together on the storage phosphor screen for autoradiography. The strips were mounted into the IPG well with molten agarose and then run at 150 V until the dye front reached the bottom of the gel. The gels were stained with Coomassie blue and dried on filter paper with a gel dryer. Similar gels were run from the cell lysates of nonradioactive cultures, and the gels were stained and stored without drying for mass spectrometry analysis.
Autoradiography.
The dried gels were exposed to a 14C-sensitive storage phosphor screen (12 x 18 cm, Super Resolution Screen; Packard Biosciences, Dowers Grove, IL) for 72 h. The exposed screens were scanned in a cyclone storage phosphor system (Packard Bioscience). The image analysis software Optiquant (Packard Bioscience) was used to quantify the radioactivity accumulation in digital light units for one-dimensional gel scans. The phosphor images of 2DGE were analyzed using KODAK image analysis software (Eastman Kodak, Rochester, NY). The analysis was performed by creating regions of interest (ROIs) around the spots generated by radioactivity on the phosphor image. ROI analysis first identifies the region/spot to be analyzed and then defines the types of analyses to be performed. The software measured the net intensity for each spot (sum of the background-subtracted pixels within the spot). The ratios for the corresponding spots between study groups (AA vs. C) were calculated from the net intensity values obtained from the ROI analysis.
Mass spectrometry.
The corresponding protein bands and spots in the gels that showed increased accumulation of radioactivity in the phosphor image were excised. The gel pieces were treated with 50% acetonitrile and 50 mM Tris, pH 8.1, for 30 min at room temperature and then reduced with 25 mM DTT and 50 mM Tris for 20 min at 55°C followed by alkylation with 45 mM iodoacetamide and 50 mM Tris for 20 min at room temperature. Overnight digestion was performed with 0.01 µg/µl trypsin (Promega, Madison WI) in 20 mM Tris, pH 8.1, at 37°C. Peptides were extracted from the gel spots with 2% formic acid, and then with 70% acetonitrile, 30% water, and 0.1% formic acid. Liquid chromatography-mass spectrometry (LC-MS)/MS analysis of the peptides was performed on a Waters Micromass Q-TOF API US quadrupole time-of-flight mass spectrometer (Waters, Milford, MA) running Masslynx 4.0. The Q-TOF, configured with the Z-spray nanoflow electron spray ionization source coupled to a Waters CapLC running a Vydac C18 150 µm x 100 mm column at 1.5 µl/min. Peptides were eluted with a gradient from 5% buffer A to 50% buffer B over 50 min, where buffer A is 0.1% formic acid, 5% acetonitrile, and 95% water and buffer B is 0.1% formic acid, 80% acetonitrile, and 5% water. The mass spectrometer experiment consists of a 1-s survey scan from 400/1,900 mass-to-charge ratio (m/z) with switching to 2-s MS/MS fragmentation scans on ions exceeding a threshold of 10 counts/s. The three most intense ions were fragmented per survey scan, and, once used, precursor ions were excluded from repeated fragmentation for 180 s. The collision energy applied to fragment the peptides was varied automatically depending on precursor m/z and charge state. The raw data were converted to PKL files, which were used to search the NCBInr protein database using local licensed Mascot (Matrix Science, London, UK) running on a 10-node cluster.
 |
RESULTS
|
---|
Tracer-specific activity.
A radioactivity count of the culture media for the C and AA groups showed that steady-state specific activity for the radioactive tracer was maintained throughout the study period. The specific activities (in µCi/mM of cold leucine) for C and for AA groups are given in Table 2, and these were not different. These specific activities were averages taken from five separate sets of experiments with duplicate samples from each well.
Gel staining vs. autoradiography.
Phosphor image scans of the electrophoresis gels showed the presence only of liver proteins newly synthesized during the study period. Figure 1 shows a Coomassie-stained gel (Fig. 1A) and phosphor image scan (Fig. 1B) of the SDS-PAGE performed on hepatocyte cell lysate proteins from both study groups. Coomassie staining shows no detectable changes in the band intensity with respect to the time of the culture as well as between C and AA. In contrast, the phosphor image scan of the same gel (Fig. 1B) clearly shows a marked and steady increase in the accumulation of radioactivity for several protein bands from corresponding 2-h to 8-h cultures. Similar phenomena were observed for the phosphor images of the 2DGE (see Fig. 3). Figure 3 shows increased accumulation of radioactivity in the protein spots as incubation time of the culture increases from 2 to 8 h, whereas the Coomassie blue staining of the respective gels was unable to show similar differences in protein spot density.

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 1. SDS-PAGE and storage phosphor scan of proteins from cell lysate of rat hepatocytes. After the experiments in culture, hepatocytes from control (C) and high amino acid (AA) media were harvested and dissolved in SDS sample buffer. Protein concentrations were determined by the Bradford method. SDS-PAGE was performed for equal amounts of proteins from cell lysate of each study group. Lanes 1, 3, and 5 are cultures from C; lanes 2, 4, and 6 are cultures from AA for 2, 4, and 8 h, respectively. Nos. on left are kDa. A: gel was stained with Coomassie blue and dried on a filter paper. There is no detectable difference in the band intensity between study groups (C vs. AA) or with respect to time. B: dried gel was exposed to storage phosphor plate sensitive to 14C for 3 days, and the plate was scanned in a Cyclone storage phosphor system to get the phosphor image scan. There is a steady increase in the radioactivity accumulation from 2-h to 8-h culture. Scan reveals that there is gradual and steady accumulation of radioactivity signal at the lowest band in the AA group compared with the C group.
|
|

View larger version (118K):
[in this window]
[in a new window]
|
Fig. 3. Two-dimensional (2-D) gel electrophoresis of cell lysate proteins from cultured rat hepatocytes and the storage phosphor image. Coomassie blue-stained gels (top) did not show any visible changes between the protein spots in the gels of proteins from C and AA. The dried 2-D gels were exposed to 14C-sensitive storage phosphor screens for 72 h. Phosphor image (bottom) shows more accumulation of radioactivity in a number of gel spots for AA than for C. This difference was more evident between C and AA in 4- and 8-h cultures than between treatment groups in 2-h culture. The figure also reveals that only selected hepatocyte cell lysate proteins are stimulated by high amino acid availability. Data shown here were reproduced in 3 separate experiments from different sets of cultures.
|
|
One-dimensional vs. 2DGE.
Phosphor image scan of the SDS-PAGE (Fig. 1B) shows increased accumulation of radioactivity for the lowest molecular mass band (indicated by an arrow) in the AA lanes compared with the C lanes at each incubation period. The data for this particular protein band are shown in Figure 2 in digital light units. This change represents an increase in the incorporation of radiolabeled leucine or synthetic rate of this liver protein. Phosphor image scan of 2DGE (Fig. 3) shows several additional protein spots with increased accumulation of radioactivity with respect to both incubation time and amino acid concentration. These proteins with differences in radioactive accumulation were not evident in the one-dimensional gel approach. Accumulation of radioactivity in the cultures of 4 and 8 h were substantially higher for these proteins in the AA than in the C group. The average fold increase in radioactivity accumulation of the spots (AA vs. C) at 4 and 8 h is given in Table 3. Although it appears to be a global effect, there are several protein spots that did not accumulate radioactivity, and several other spots show no change in the radioactivity accumulation between the C and AA groups (signal intensity ratio of 1). Therefore, during the experimental period, several proteins showed no synthesis, and several other proteins showed synthesis but then no detectable differences of the increment in radioactivity between the two experimental conditions (AA vs. C). Only the spots that consistently showed a difference of
1.4 (signal intensity ratio of AA vs. C) are reported. These spots showed a wide range of differences in radioactivity (Table 4). Thus 17 spots were selected for protein identification by tandem mass spectrometry. A signal intensity ratio of
1.4 was considered significant because, on triplicate experiments, all of these 17 spots showed a consistent ratio of
1.4.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2. Analysis of storage phosphor image. Graphic representation of radioactive signal accumulation of the lowest band from Fig. 1B. Measurement is expressed in digital light units (DLU) by OptiQuant software provided in the Cyclone storage phosphor system. The protein from this band is identified as chaperonin-10 by tandem mass spectrometry. Band shows gradual and steady accumulation of radioactivity from 2 to 8 h and enhanced signal in AA compared with C. Data shown here were from 5 separate experiments.
|
|
View this table:
[in this window]
[in a new window]
|
Table 3. Average fold change of radioactivity accumulation (signal intensity ratio) of spots in AA vs. Control groups at 4 and 8 h from 3 separate cultures
|
|
Proteins whose synthetic rates are stimulated by high amino acid concentration.
The protein in Fig. 2, which was separated by SDS-PAGE and is shown to have increased radioactivity accumulation by phospor image, was identified as chaperonin-10. Fifteen other liver proteins were identified by tandem mass spectrometry from 17 2DGE-gel spots (Fig. 4) that showed increased accumulation of radioactivity in the AA group compared with the C group. Therefore, a total of 16 proteins were identified that have a consistently higher (
1.4) signal intensity ratio (AA vs. C). The proteins identified are shown in Table 4 and their functions indicated.

View larger version (130K):
[in this window]
[in a new window]
|
Fig. 4. Identification of proteins whose synthetic rate was increased by high amino acid availability. A: Coomassie blue-stained gel; B: phosphor image scan of the dried gel. 2-D gel of cell lysate proteins from cultured rat hepatocytes was stained with Coomassie blue. Arrows and numbers indicate protein spots that have increased accumulation of radioactivity during culture in the presence of high amino acid concentration. Only the protein spots that show consistent results from 3 separate experiments were selected. Proteins from these numbered spots were identified by tandem mass spectrometry.
|
|
 |
DISCUSSION
|
---|
We describe an approach that allows simultaneous measurement of the incorporation of radiolabeled leucine into multiple specific proteins in a primary liver cell culture. This technique was applied to identify proteins whose synthetic rates were stimulated by increased amino acid supply. We identified 16 specific liver proteins whose synthetic rates are stimulated by amino acid supplementation. During the short span of the study period, no demonstrable quantitative changes in these proteins were observed. However, the use of radiolabeled leucine demonstrated an increase in the incorporation of leucine (radioactivity) in 16 specific proteins. The combination of labeled amino acid tracer with the current proteomic approaches for purification and identification of specific proteins from a mixture allowed identification of these liver proteins, which are regulated by amino acids.
To our knowledge, the technique described has been applied for the first time to determine simultaneously several liver proteins whose synthetic rates are enhanced by amino acids. There may be several other proteins whose synthetic rates are stimulated by amino acids but not detected by the current approach. There are many potential explanations for why only a limited number of proteins among thousands of proteins were identified with increased synthetic rates with amino acid supplementation. It is likely that most of the proteins' maximal capacity for synthesis was already achieved with the standard amino acid concentration in the media; therefore, increasing the amino acid concentration in the media had no additional effect. There may be many other proteins whose synthetic rates increased less than 1.4-fold in response to high amino acid concentration and were therefore not considered for the current study. A more sensitive technique or techniques than this seminal attempt may be needed to detect the proteins with low synthetic rates and concentration. Moreover, the 2DGE system that we used does not separate alkaline proteins. Previous studies of metabolic labeling with radiolabeled amino acids and 2DGE were performed in yeast (23) or bacteria (3) to quantify proteins. The primary liver cultures were chosen for the current experiment because they offer a potential opportunity to expose liver cells in vivo to different physiological states (e.g., starvation or overfeeding) and compare responses to interventions in vitro.
The method described in the present study is an innovation of a previously reported technique (23) for two-dimensional comparison of proteins. One distinction is that we used 2DGE for each study condition separately instead of using two radioisotopes and coelectrophoresis of two samples as in a previous report (23). By doing 2DGE on both of the study groups separately, we avoided using radioisotopes of different energy levels, two types of storage phosphor plates, time-consuming successive phosphor image scanning of the coelectrophoresis gel, and the need for 14C-to-3H ratio measurement. Moreover, selection of 14C as the radioisotope label allowed us to use dried gels for direct exposure to storage phosphor plates and to detect proteins having very low molecular weights. It would be necessary to transfer proteins to nitrocellulose membranes when 3H label was used to avoid contamination of the 3H-sensitive phosphor plates because of the lack of protective covering on the 3H-sensitive phosphor plates. Furthermore, we could expose the dried gels from the study groups simultaneously on the same phosphor plate for comparable data. Leucine was chosen as the labeling amino acid in the current experiment because it is one of the most abundant amino acids in body proteins, because it is readily available, and because of the absence of detectable interconversion of leucine into another amino acid during in vivo labeling (1, 6, 25, 27); it thus offers opportunities for future in vivo studies. The effect of l-[U-14C]leucine on hepatocytes should be negligible, since we added only 1 µCi/mM cold leucine (specific activity 277 mCi/mM) present in the medium.
We opted for a balanced mixture of all amino acids rather than individual amino acids to investigate the effect of amino acid availability on liver protein synthesis. A balanced mixture of amino acids is expected to better maintain protein synthesis and is more physiologically relevant. A recent study in rats (2) showed stimulation of ribosomal protein mRNA translation by leucine but no concomitant enhancement in the total liver protein synthesis. This suggests that a complement of all amino acids is required for the activation of signaling pathways involved in liver protein synthesis. It has been demonstrated that deprivation of a single amino acid such as histidine (21) or a mixture of essential amino acids like tryptophan, leucine, and isoleucine (15) from culture medium of rat hepatocytes suppressed albumin synthesis and total protein synthesis. These studies show only a marginal effect on total protein synthesis compared with the synthesis of albumin when the culture medium was deprived of individual amino acids.
As another important methodological aspect of the current study, we compared standard and high amino acid concentrations in the culture media. Increased synthesis of plasma proteins like albumin has been observed in cultured rat hepatocytes when amino acid concentrations in culture media were increased, even up to portal levels (24). Such an increase in protein synthesis has been observed in the splanchnic bed of healthy human subjects when they received an amino acid infusion at levels threefold above baseline (26). The present study is comparable to the previous in vivo studies. The novel finding from the present study is that an amino acid level comparable to what is found in postprandial portal blood caused the stimulation of synthesis of several specific liver proteins.
The proteins identified from 2DGE spots are tabulated (Table 4) in descending order for their signal intensity ratio in AA media to C media. Phosphor images of the one-dimensional gel electrophoresis showed only chaperonin-10, with a considerable difference in signal intensity between the AA and C groups. Our results clearly show the superiority of 2DGE coupled with the phosphor image approach to identify liver proteins whose synthetic rates are stimulated by amino acids.
Among the proteins whose synthetic rates were enhanced by amino acids, we identified three proteins involved in protein synthesis such as peptide chain initiation [eukaryotic translation initiation factor 5A (eIF5A)], protein folding (chaperonin-10), and protein modification and proteolysis (ubiquitin). The protein present in the gel band of Fig. 2 was identified as chaperonin-10 and is also known as heat shock protein (Hsp)-10. This chaperone serves as a cochaperone for Hsp-60 in the protein folding and assembly processes and is found in organelles such as mitochondria (12). The detection of enhanced synthesis of chaperonin-10 in high-amino acid cultures is important, because it is believed that these chaperonins are essential for transfer of newly synthesized protein chains between chaperones and to prevent protein aggregation (9). The initiation of mRNA translation in eukaryotes requires several multi-subunit complexes known as eIFs. Protein synthesis is inhibited at the level of peptide chain initiation due to decreased activity of eIF2B by deprivation of the essential amino acid histidine in perfused rat liver (20). In vivo study in rats showed that oral administration of leucine stimulates the ribosomal protein mRNA translation by hyperphosphorylation of eIF4E-binding protein that in turn allows the assembly of eIF4F complex (2). Our results identified increased synthesis of eIF5A in hepatocyte cultures with high amino acid concentration. eIF5A is a ubiquitous protein and is one of at least five or six factors necessary for the initiation of eukaryotic cellular protein biosynthesis and thought to be necessary for selective mRNA stabilization and translation (18, 19). Ubiquitin is a compact globular protein involved in the ubiquitin-proteosome proteolytic pathway, one of the two major routes of protein degradation (7). The effect of amino acids on ubiquitin synthesis in mammalian liver cells was previously unknown, although inhibition of ATP-ubiquitin-dependent proteolysis in rat skeletal muscle by branched-chain amino acids has been reported (5). It is likely that, in all conditions when protein synthesis is increased, a proportional increase in protein breakdown (proteolysis) is needed to dispose of the protein after its metabolic action. Besides chaperonin-10, the mass spectrometry analysis of the particular gel band identified protein-binding transcriptional activator (PBTA). On the basis of the score (50 for chaperonin-10 vs. 20 for PBTA) and the number of unique peptides identified (13 vs. 3), we consider chaperonin-10 the most abundant protein of the gel band. However, we cannot exclude at this point that chaperonin-10 is the only protein in this band that is taking up the label. This may represent a limitation of one-dimensional gel electrophoresis in resolving individual proteins. Of note, the two proteins shown in this gel band are involved in protein synthesis.
The current study shows that the synthetic rates of a number of enzymes are increased by amino acids in liver cells. They include cytochrome c oxidase Va (COX), ATP synthase
-chain, glutathione S-transferase (GST), catechol O-methyl transferase (COMT), and arginase-1. COX and ATP synthase
-chain are enzymes involved in energy metabolism. The effect of amino acids on COX and ATP synthase has not been reported previously. These findings support the recent report of increased ATP production in skeletal muscle when the amino acid mixture was infused with insulin (28). There was also an associated increase of COX enzyme activity and transcript level of COX-3. Increased mitochondrial biogenesis allows an increase in the availability of ATP for energy-consuming protein synthesis. In addition to these two enzymes, apocytochrome b5, an electron transfer protein involved in methemoglobin reduction, was also increased by amino acids in the current investigation.
We also noted that synthesis of COMT and GST was increased more than twofold in hepatocyte cultures during high amino acid availability. COMT catalyzes the O-methylation of biologically active or toxic catechols and has a crucial role in the metabolism of drugs and neurotransmitters such as L-dopa, norepinephrine, epinephrine, and dopamine. GST catalyzes the addition of the tripeptide glutathione to endogenous and xenobiotic substrates possessing electrophilic functional groups. We observed amino acid-induced stimulation of prohaptoglobin in this study. Haptoglobin is an acute-phase protein that acts as an antioxidant by virtue of its ability to prevent hemoglobin-driven oxidative tissue damage (22). Our result is consistent with the results of previous studies using a murine model (14) as well as perfused rat liver (17). A key enzyme in the urea cycle, arginase-1, was also found to be stimulated in liver cells by amino acids in the current study. Arginase catalyzes the hydrolysis of arginine to ornithine and urea. The detection of increased synthesis of arginase is in agreement with a previous observation of increased activity of this enzyme when rat hepatocytes were treated with amino acid supplementation (32).
Increased amino acid levels stimulated many other liver proteins.
2-U-globulin is a major protein in rat urine having binding and transport properties. Membrane-associated progesterone receptor component is a protein expressed predominantly in the liver and kidney and mediates progesterone action. Structural proteins like actin and keratin were also found to have enhanced synthetic rates upon amino acid treatment. Synthesis of
-actin (actin cytoplasmic 2) was found enhanced during the high amino acid availability. Cytoplasmic actin filaments play an important role in the maintenance of cell shape, division, and motility. It is reported that
-actin protein synthesis is enhanced in regenerating rat liver (29). Other structural proteins, keratin type II cytoskeletal-1 and keratin type II cytoskeletal-8, were increased in the cultures of the AA group. Analysis of the mass spectral data matched peptides to the cytokeratins of mouse and rat, respectively. Although we do not have any supporting reports for the increased synthesis of these keratins in liver cells in response to amino acid treatment, an increased expression of keratin polypeptides in long-term culture of adult rat hepatocytes was reported previously (11). We also observed increased synthesis of albumin in cultures with high concentrations of amino acids. The present results are consistent with previous reports that albumin synthesis is increased in liver cells by amino acids (15, 16) and decreased during amino acid deprivation (10).
The current study has demonstrated that synthesis rates of several liver proteins can be measured simultaneously by combining metabolic labeling of proteins, purification by 2DGE, and tandem mass spectrometry. We have discussed the limitations and the potential scope of this approach. Applying this novel approach, we have demonstrated that increased amino acid concentration in culture media of hepatocytes has a stimulatory effect on the rate of synthesis of 16 specific liver proteins. The proteins profiled and identified are involved in different and specific functions, including protein synthesis, protein folding and modification, oxidative phosphorylation, antioxidant effects, signal transduction, and transport, as well as cell motility and structural integrity.
 |
GRANTS
|
---|
This study was supported by a grant from the National Institutes of Health RO-1 AG-409531 to David Murdock-Dole Professorship (K. S. Nair), and the Mayo Foundation.
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge the skillful technical support of Benjamin Madden of Mayo Proteomics Research Center in mass spectrometry, the critical comments of our colleagues Kevin Short, Yan Asmann, Paul Rys, and Katherine Klaus, and the secretarial support of Melissa Aakre.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: K. S. Nair, Endocrinology Research Unit, Mayo Clinic, Rochester, Minnesota 55905 (E-mail: nair{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
 |
REFERENCES
|
---|
- Ahlman B, Charlton M, Fu A, Berg C, O'Brien P, and Nair KS. Insulin's effect on synthesis rates of liver proteins. A swine model comparing various precursors of protein synthesis. Diabetes 50: 947954, 2001.[Abstract/Free Full Text]
- Anthony TG, Anthony JC, Yoshizawa F, Kimball SR, and Jefferson LS. Oral administration of leucine stimulates ribosomal protein mRNA translation but not global rates of protein synthesis in the liver of rats. J Nutr 131: 11711176, 2001.[Abstract/Free Full Text]
- Bernhardt J, Buttner K, Scharf C, and Hecker M. Dual channel imaging of two-dimensional electropherograms in Bacillus subtilis. Electrophoresis 20: 22252240, 1999.[CrossRef][ISI][Medline]
- Berry MNEA and Barritt GJ. Isolated Hepatocytes: Preparation, Properties and Applications. New York: Elsevier, 1991.
- Busquets S, Alvarez B, Llovera M, Agell N, Lopez-Soriano FJ, and Argiles JM. Branched-chain amino acids inhibit proteolysis in rat skeletal muscle: mechanisms involved. J Cell Physiol 184: 380384, 2000.[CrossRef][ISI][Medline]
- Charlton M, Ahlman B, and Nair KS. The effect of insulin on human small intestinal mucosal protein synthesis. Gastroenterology 118: 299306, 2000.[ISI][Medline]
- Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell 79: 1321, 1994.[ISI][Medline]
- De Feo P, Gaisano MG, and Haymond MW. Differential effects of insulin deficiency on albumin and fibrinogen synthesis in humans. J Clin Invest 88: 833840, 1991.[ISI][Medline]
- Ellis RJ and Hartl FU. Principles of protein folding in the cellular environment. Curr Opin Struct Biol 9: 102110, 1999.[CrossRef][Medline]
- Flaim KE, Liao WS, Peavy DE, Taylor JM, and Jefferson LS. The role of amino acids in the regulation of protein synthesis in perfused rat liver. II. Effects of amino acid deficiency on peptide chain initiation, polysomal aggregation, and distribution of albumin mRNA. J Biol Chem 257: 29392946, 1982.[Abstract/Free Full Text]
- Gupta PD and Bhonde RR. Increased expression of keratin polypeptides in long term culture of adult rat hepatocytes. Cytobios 70: 123130, 1992.[Medline]
- Gupta RS. Evolution of the chaperonin families (Hsp60, Hsp10 and Tcp-1) of proteins and the origin of eukaryotic cells. Mol Microbiol 15: 111, 1995.[ISI][Medline]
- Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, and Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17: 994999, 1999.[CrossRef][ISI][Medline]
- Hegyi K, Fulop AK, Toth S, Buzas E, Watanabe T, Ohtsu H, Ichikawa A, Nagy A, and Falus A. Histamine deficiency suppresses murine haptoglobin production and modifies hepatic protein tyrosine phosphorylation. Cell Mol Life Sci 58: 850854, 2001.[ISI][Medline]
- Hutson SM, Stinson-Fisher C, Shiman R, and Jefferson LS. Regulation of albumin synthesis by hormones and amino acids in primary cultures of rat hepatocytes. Am J Physiol Endocrinol Metab 252: E291E298, 1987.[Abstract]
- Ijichi C, Matsumura T, Tsuji T, and Eto Y. Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system. Biochem Biophys Res Commun 303: 5964, 2003.[CrossRef][ISI][Medline]
- John DW and Miller LL. Regulation of net biosynthesis of serum albumin and acute phase plasma proteins. Induction of enhanced net synthesis of fibrinogen, alpha1-acid glycoprotein, alpha2 (acute phase)-globulin, and haptoglobin by amino acids and hormones during perfusion of the isolated normal rat liver. J Biol Chem 244: 61346142, 1969.[Abstract/Free Full Text]
- Kang HA and Hershey JW. Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J Biol Chem 269: 39343940, 1994.[Abstract/Free Full Text]
- Kim KK, Hung LW, Yokota H, Kim R, and Kim SH. Crystal structures of eukaryotic translation initiation factor 5A from Methanococcus jannaschii at 1.8 A resolution. Proc Natl Acad Sci USA 95: 1041910424, 1998.[Abstract/Free Full Text]
- Kimball SR, Antonetti DA, Brawley RM, and Jefferson LS. Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 alpha phosphatase activity. J Biol Chem 266: 19691976, 1991.[Abstract/Free Full Text]
- Kimball SR, Yancisin M, Horetsky RL, and Jefferson LS. Translational and pretranslational regulation of protein synthesis by amino acid availability in primary cultures of rat hepatocytes. Int J Biochem Cell Biol 28: 285294, 1996.[CrossRef][ISI][Medline]
- Langlois MR and Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 42: 15891600, 1996.[Abstract/Free Full Text]
- Monribot-Espagne C and Boucherie H. Differential gel exposure, a new methodology for the two-dimensional comparison of protein samples. Proteomics 2: 229240, 2002.[CrossRef][ISI][Medline]
- Montoya A, Gomez-Lechon MJ, and Castell JV. Influence of branched-chain amino acid composition of culture media on the synthesis of plasma proteins by serum-free cultured rat hepatocytes. In Vitro Cell Devel Biol 25: 358364, 1989.[ISI][Medline]
- Nair KS, Ford GC, Ekberg K, Fernqvist-Forbes E, and Wahren J. Protein dynamics in whole body and in splanchnic and leg tissues in type I diabetic patients. J Clin Invest 95: 29262937, 1995.[ISI][Medline]
- Nygren J and Nair KS. Differential regulation of protein dynamics in splanchnic and skeletal muscle beds by insulin and amino acids in healthy human subjects. Diabetes 52: 13771385, 2003.[Abstract/Free Full Text]
- Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, and Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1: 376386, 2002.[Abstract/Free Full Text]
- Stump CS, Short KR, Bigelow ML, Schimke JM, and Nair KS. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts. Proc Natl Acad Sci USA 100: 79968001, 2003.[Abstract/Free Full Text]
- Tanahashi T, Suzuki M, Itoh N, and Mitsui Y. Enhancement of gamma-actin protein during liver regeneration: its accumulation in a region adjacent to the hepatocyte plasma membrane. J Biochem (Tokyo) 118: 355363, 1995.[Abstract]
- Unlu M, Morgan ME, and Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18: 20712077, 1997.[ISI][Medline]
- Volpi E, Lucidi P, Cruciani G, Monacchia F, Reboldi G, Brunetti P, Bolli GB, and De Feo P. Contribution of amino acids and insulin to protein anabolism during meal absorption. Diabetes 45: 12451252, 1996.[Abstract]
- Washizu J, Chan C, Berthiaume F, Tompkins RG, Toner M, and Yarmush ML. Amino acid supplementation improves cell-specific functions of the rat hepatocytes exposed to human plasma. Tissue Eng 6: 497504, 2000.[CrossRef][ISI][Medline]
- Yoshizawa F, Kimball SR, Vary TC, and Jefferson LS. Effect of dietary protein on translation initiation in rat skeletal muscle and liver. Am J Physiol Endocrinol Metab 275: E814E820, 1998.[Abstract/Free Full Text]