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-Aminoadipate 
-Semialdehyde Synthase mRNA Knockdown Reduces the Lysine Requirement of a Mouse Hepatic Cell Line1–3,
Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506
* To whom correspondence should be addressed. E-mail: kbleming{at}wvu.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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-Aminoadipate 
-semialdehyde synthase (AASS) is the bifunctional enzyme containing the lysine -ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first 2 steps in the irreversible catabolism of lysine. A rare disease in humans, familial hyperlysinemia, can be caused by very low LKR activity and, as expected, reduces the lysine "requirement" of the individual. This concept was applied to a murine hepatic cell line (ATCC, FL83B) utilizing RNA interference (RNAi) to achieve AASS mRNA knockdown. Cells were antibiotic selected for stable transfection of 2 plasmids that express different short hairpin RNA sequences for AASS knockdown. Compared with the wild-type cell line, AASS mRNA abundance was reduced 79.0 ± 6.4% (P < 0.05), resulting in a 29.8 ± 5.2% (P < 0.05) reduction in AASS protein abundance, 41.3 ± 10.0% (P < 0.05) less LKR activity, and a reduction in lysine oxidation by 50.7 ± 11.8%. To determine the effect of AASS knockdown on the lysine requirement, cells were grown in media containing 12.5, 25.0, 50.0, 100, or 200 µmol/L lysine. Using a segmented model approach for growth rate analysis, the lysine requirement of the cell line with AASS silencing was 43.4 ± 1.7 µmol/L, 
26% lower (P < 0.05), than the lysine requirement of the wild-type cell line. These results indicate AASS knockdown decreases the lysine requirement of the cell via a reduction of lysine catabolism through the saccharopine pathway, providing the initial proof in principle that RNAi can be used to reduce the nutrient requirement of a system.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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To improve lysine nutriture, a clear understanding of lysine catabolism and its regulation will be helpful. The main pathway for the irreversible degradation of lysine is catalyzed by the enzymes lysine -ketoglutarate reductase (LKR)6 (EC 1.5.1.8.) and saccharopine dehydrogenase (EC 1.5.1.9), which are located on the bifunctional protein, -aminoadipate -semialdehyde synthase (AASS) (9). Hyperlysinemia, a rare inherited disorder in humans, can be caused by a defective AASS enzyme. This disease is characterized by a significant reduction in lysine degradation through the AASS pathway that is diagnosed by detecting a reduced LKR and saccharopine dehydrogenase activity or slower lysine oxidation (LOX) (10–12). As a result of a lower oxidative flux of lysine through the saccharopine pathway, lysine accumulates in the blood to a level 10 times higher than the normal value (11). Individuals with familial hyperlysinemia often have motor and mental handicaps that tend to advance with age (12). Hyperlysinemic children who consume a diet that would ordinarily be deficient in lysine exhibit a decline in plasma lysine concentrations to near-normal levels and under these conditions both growth and development improve (13). Interestingly, this indicates that individuals with familial hyperlysinemia have a lower lysine requirement that is caused by reduced lysine catabolism through the AASS pathway.
Despite the symptoms, familial hyperlysinemia represents a condition in which the body uses lysine more efficiently by significantly reducing lysine degradation. The application of this condition to production animals would provide for considerable economic savings, because feed can be reformulated to contain less protein to meet a lower lysine requirement. Reducing dietary protein also means feeding less excess amino acids, subsequently reducing the nutrient content of animal manure and attenuating the environmental impact of farming. In 1995, the cost of adding synthetic lysine to swine diets accounted for 1.5% of total feed costs (14), which translates to 100 million dollars per year that swine producers in a farrow-to-finish operation spend on feed-grade lysine. Therefore, the swine industry is particularly poised to benefit considerably from the reduction of animals' lysine requirement.
The long-term goal of this research is to lower the lysine requirement of animals. As a step toward this, our objective in this study was to reduce the lysine requirement of a murine hepatic cell line by utilizing RNA interference (RNAi) to decrease AASS mRNA.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Two plasmids, pSilencer4.1-CMVneo and pSilencer4.1-CMVpuro (Ambion), were selected for stable expression of short hairpin RNA (shRNA) sequences in mammalian cells while conferring resistance to different antibiotics. Sequences used for RNAi were generated against mouse AASS using the small-interfering RNA Selection Server (15). The following 4 sense sequences were chosen: 1) 5'-AUGUCCUGAAUUACCACAC-3'; 2) 5'-AUCUUGUGAUCAGCUUGUU-3'; 3) 5'-UCACUGCAAGCTACAUUAC-3'; and 4) 5'-ACUCAUCAACAGAGAAGCA-3'. The loop sequence, CUUGCUUC, joined the sense and antisense sequences in the hairpin structure. Oligonucleotides coding for the shRNA sense and antisense strands were generated with BamHI and HindIII restriction sites flanking the shRNA to facilitate ligation into the pSilencer4.1-CMVneo and pSilencer4.1-CMVpuro plasmids. Plasmid sequences were verified by direct sequencing. An additional nonsense shRNA sequence that was designed by Ambion to have limited similarity to the mouse genome database served as a negative control and was ligated into pSilencer4.1-CMVneo and pSilencer4.1-CMVpuro vectors.
Cell culture and transfection conditions
A mouse hepatic cell line was purchased from ATCC (FL38B: CRL-2390) and grown in F-12K medium (ATCC) supplemented with 10% fetal bovine serum (v:v), 500,000 international units/L penicillin, and 0.5 g/L streptomycin.
Initial transfection. On d 1, 2.4 x 104 cells/well were seeded in a 24-well plate. On d 2, immediately before transfection, growth media was replaced with antibiotic-free growth media, 0.4 µg pSilencer4.1-CMVneo, and 1 µL Lipofectamine2000 (Invitrogen) per transfection reagent protocol. The transfection was completed for each of the 4 shRNA sequences for AASS knockdown and the nonsense shRNA sequence. Six hours post-transfection, the media was replaced with penicillin/streptomycin containing growth media. Twenty-four h post-transfection, cells were expanded to a 60-mm cell culture plate with growth media supplemented with 0.9 g/L G418. Surviving colonies were selected and expanded. Cells expressing shRNA sequences for AASS were named for the respective shRNA sequence they expressed and were indicated as shRNA1, shRNA2, shRNA3, or shRNA4. Cells that expressed the nonsense shRNA sequence were indicated as shRNAN.
Double transfection. The nonsense sequence and the shRNA sequence that yielded the second highest AASS mRNA knockdown (5'-AUGUCCUGAAUUACCACAC-3') were ligated into pSilencer4.1-CMVpuro vectors. The pSilencer4.1-CMVpuro vector with the nonsense shRNA sequence was transfected into cells from the initial transfection that expressed the nonsense shRNA from the pSilencer4.1-CMVneo plasmid. The pSilencer4.1-CMVpuro with the AASS shRNA sequence was transfected into the cell line with the highest amount of AASS knockdown from the initial transfection. The conditions for the second transfection were as follows: 6.0 x 104 cells per well were plated with antibiotic-free growth media on a 24-well plate with 48.5 µL Opti-MEM media (Invitrogen), 1.5 µL XP-sport (Ambion), and 0.5 µg pSilencer4.1-CMVpuro with either the shRNA to AASS or the nonsense sequence per transfection reagent protocol. Antibiotic selection and colony expansion proceeded as above with 450 µg/L puromycin media. Three cell lines that were used in subsequent experiments were the wild-type cell line, the double-nonsense transfected cell line (shRNANN), and the cell line that exhibited the greatest AASS knockdown after the second transfection (shRNA335).
Real-time RT-PCR
We used real-time RT-PCR to analyze mRNA knockdown in cell lines using acidic ribosomal protein (ARP) as a reference gene. A T-75 flask of cells was washed with 5 mL of cold Hank's balanced salts solution and layered with 3 mL Trizol LS (Invitrogen) for collection of RNA per the manufacturer's instructions. RNA was quantified and the quality was estimated using the OD260:OD280 ratio. Two µg of RNA was DNase (Promega) treated and reverse transcribed using random primers (Invitrogen) and Moloney murine leukemia virus (Promega) per the manufacturer's protocol. Complementary DNA was diluted 1:4 with nuclease free water and 2 µL was used in a 20-µL PCR with 10 µL 2x SYBR Green Supermix (Bio-Rad), 1.25 µmol/L forward ARP primer (5'-CAACCCAGCTCTGGAGAAAC-3'), and 1.25 µmol/L reverse ARP primer (5'-GTGAGGTCCTCCTTGGTGAA-3') or 0.625 µmol/L forward AASS primer (5'-TGGAGACTTCAACGGCTTCT-3') and 0.625 µmol/L reverse AASS primer (5'-TGGCCCATAGATCTCCTTTG-3'). The real-time PCR was performed on a BioRad iCycler IQ detection system using a 3-step touch-down procedure. The procedure began with a "hot-start" at 95°C for 5 min, followed by a cycle of 95°C denaturing for 15 s, 70°C annealing for 30 s, and 72°C extension for 30 s. The next 4 PCR cycles followed the same temperature sequence and time lengths except the annealing temperature was reduced by 1°C with each subsequent cycle. The final 35 PCR cycles proceeded at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Melt curve analysis confirmed a single PCR product in each reaction. The products were sequenced and confirmed as AASS or ARP. Real-time PCR data were analyzed using the efficiency corrected relative expression method (16).
Western blotting
Cells were harvested from flasks using trypsin and 3 x 106 cells were pelleted by centrifugation at 300 x g; 10 min at 4°C. The cell pellet was resuspended in 100 µL lysis buffer (20 mmol/L HEPES, 10 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 50 mmol/L β-glycerophosphate, 5 mmol/L EDTA, 1 mmol/L sodium orthovanadate, 2 mmol/L benzamidine, 100 µmol/L leupeptin, 100 µmol/L pepstatin, 250 µmol/L soybean trypsin inhibitor, 200 µmol/L phenylmethylsulfonyl fluoride, 0.50% Triton-X100, pH 7.4) and incubated for 1 h at 4°C on a Nutator. The lysed cells were centrifuged at 300 x g for 10 min and the cell lysate was removed for western blotting. Analysis of AASS abundance was based on an equal number of cells and not per microgram protein. Therefore, only the protein concentration of the wild-type lysate was determined using Coomassie Plus Protein Assay reagent (Pierce Biotechnology) with bovine serum albumin as a standard. SDS-PAGE and western blotting were performed using a published protocol (17) with 15 µg wild-type cell lysate protein and an equal volume of shRNANN and shRNA335 cell lysate (i.e. an equal number of cells). Primary antibodies were generated in rabbits (Invitrogen) to a 15-amino acid residue of mouse AASS. The secondary antibody was goat anti-rabbit conjugated to horseradish peroxidase and detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). The band intensity was quantified using densitometry (Flourochem 8000, Alpha Innotek).
Lysine -ketoglutarate reductase assay
LKR activity was determined on mitochondria isolated from individual cell lines using a modification of an established procedure (18) with a SpectraMax Plus384 microplate reader (Molecular Devices). To isolate mitochondria, 4.0 x 107 cells were lifted from flasks with trypsin and collected at 500 x g; 5 min at 4°C. Cells were resuspended with ice-cold homogenization buffer (200 mmol/L mannitol, 70 mmol/L sucrose, 5 mmol/L HEPES, 1 mmol/L EGTA, 0.5 g/L bovine serum albumin, 5 mmol/L 2-mercaptoethanol, pH 7.4) and homogenized with 70 strokes using a small clearance pestle. The homogenate was centrifuged at 3000 x g; 5 min. The supernatant was collected, centrifuged at 10,000 x g; 5 min, and the pellet containing mitochondria was resuspended in 60 µL homogenization buffer.
LOX assay
LOX was measured with slight modifications to a previously described procedure (18). Lysine 14C-labeled only at the 1 carbon (L-[1-14C], American Radiolabeled Chemicals), a generous gift from Dr. Norlin Benevenga (University of Wisconsin, Madison), was purified using a cation exchange Dowex-50 column. Before correcting for the percent recovery, which was typically 85–95%, the specific activity of the lysine was 2.368 kBq/nmol. One mL of LOX buffer (0.1 mmol/L L-lysine, 314 mmol/L mannitol, 78 mmol/L sucrose, 20 mmol/L HEPES, 6 mmol/L Mg2Cl, 0.4 mmol/L EDTA, 30 mmol/L -ketoglutarate, and 1 mmol/L NADPH, pH 7.0) was combined with 3.5 x 106 cells resuspended in 1 mL homogenization buffer in a 25-mL Erlenmeyer flask.
Determination of the lysine requirement
Media containing 12.5, 25.0, 50.0, 100, or 200 µmol/L lysine supplemented with 10% dialyzed fetal bovine serum (v:v) and antibiotics was created by mixing F-12K media lacking lysine (SAFC Biosciences) with unmodified F-12K media (ATCC). On d 0, 6.0 x 104 cells were seeded with treatment media in each of 4 wells on a 12-well plate with 1 plate dedicated to each cell line by treatment combination. Cells in 1 well from each plate were counted using a hemacytometer every 12 h after the initial seeding for 48 h. Growth rates were determined by plotting the hours after the initial seed vs. the log (cell count) and generating a line of best fit. The slope of the line represented the growth rate of the cells for each cell line by treatment combination. The maximal growth rate was determined by averaging the growth rate of the cells treated with media containing lysine concentrations higher than the requirement of the cell.
Statistics
Data were analyzed by ANOVA with the PC-SAS general linear models procedure for significant differences between treatment means. In the event of a significant F-value, the least significant difference procedure was used for means comparisons. To determine the lysine requirement of cells, cell growth rates were fitted to an NLIN SAS segmented model curve. The intersection of the linear segments estimates the lysine requirement of the cell line. A bootstrap analysis of replicates and subsequent ANOVA and least significant difference procedure was completed to determine differences in the lysine requirement and growth rates between cell lines.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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30% and LKR activity and LOX were reduced to almost 50% of wild-type levels. As a result of less LOX and not a slower growth rate, the lysine requirement of the cells exhibiting AASS silencing was reduced 26% compared with wild-type cells. Therefore, this study demonstrates that RNAi can be used to modify the nutrient requirements of a system. More specifically, these data indicate the potential for AASS silencing as a mechanism for improving the efficiency of lysine utilization for protein synthesis in livestock, especially swine and poultry, in which lysine is a first and second limiting amino acid, respectively. Despite the large reduction in AASS mRNA and protein abundance, LKR activity and LOX did not decrease to numerically comparable levels in shRNA335 cells. Although this is surprising, previous research in plants indicates that post-translational modifications to the AASS protein are primarily responsible for changes in its activity. In tobacco seeds, regulation of LKR activity operates through protein phosphorylation and intracellular signaling cascades with calcium as a second messenger (19). Research in soybeans indicates that LKR activity decreases with dephosphorylation of the AASS enzyme (20), lending further support for the proposed role of post-translational modifications as a regulator of AASS activity. In animals, it is unknown if AASS phosphorylation occurs, but in fish (21), diet-mediated changes in AASS mRNA and LKR activity suggest post-translational modifications as a mechanism for regulation of AASS activity. In the present experiment, it is not likely that there are differences in the post-translational modification of AASS between wild-type cells and cells with AASS knockdown. Rather, it is suggested that the drastic reduction in AASS mRNA abundance in shRNA335 cells was sufficient to overcome the endogenous post-translational regulation of AASS and result in a reduction in AASS protein abundance and lysine catabolism.
The assay measuring LKR activity was completed at 50 mmol/L lysine, which is well into Vmax conditions, whereas the LOX assay was conducted at 50 µmol/L lysine, which is less than the Km for LKR. Therefore, to compare the rates of lysine catabolism between the 2 assays, the Michaelis-Menton equation and an experimentally determined Km of 15.5 mmol/L lysine (data not shown) were utilized to scale the rate of LKR activity down to 50 µmol/L lysine, followed by correction for mitochondrial recovery during sample preparation, which was typically 40%. After these calculations, the LKR activity, in fkat/106 cells in 50 µmol/L lysine, was 0.885 for wild-type cells, 0.938 for shRNANN cells, and 0.507 for shRNA335 cells. With equal units employed, the rate of lysine catabolism in the LOX assay was about twice that determined from the LKR assay. A previous report (18) indicated an opposite and greater difference between these 2 values in rat liver, citing the LKR activity rate as being 6- to 100-fold greater than LOX. Differences between cell culture and whole animals, assay conditions, and sample preparation may have contributed to the difference in the 2 observations.
The lysine requirement of the cell lines was determined using SAS NLIN to fit growth rates to a 2 straight-line, 1-breakpoint model in which the lysine concentration where the breakpoint occurred estimates the lysine requirement. An additional model that was considered for use was the quadratic broken-line model that fits data below the requirement to a quadratic curve, rather than a straight line, which tends to estimate a higher requirement than straight-line 1-breakpoint models (22). Data were fit to both models and although the quadratic broken-line model gave lysine requirements numerically higher (data not shown) than the 2 straight-line, 1-breakpoint model, both models found the shRNA335 cell line had a significantly lower lysine requirement.
Because reducing the AASS mRNA abundance decreased the lysine requirement in cell culture, the economic potential for the incorporation of AASS knockdown into livestock production becomes conceptually feasible. In the present experiment, AASS knockdown to 20% of wild-type mRNA levels resulted in only a 50% reduction in LKR activity and LOX compared with 
90% reduction in LKR activity and LOX seen in humans with familial hyperlysinemia. Short interfering RNA sequences silence genes with varying efficacy, which provides an element of control over the extent to which AASS mRNA or activity is reduced. Therefore, in livestock, the increase in the plasma lysine concentration would likely be less severe with AASS mRNA knockdown and the developmental phenotypes observed in humans with familial hyperlysinemia would likely not occur. Additionally, the objective behind AASS knockdown in livestock is to reduce the lysine requirement, with the rationale that reducing the lysine requirement permits the consumption of a cheaper, low-lysine diet, which would prevent the onset of hyperlysinemia.
Reducing the lysine requirement in poultry and swine by utilizing AASS RNAi seems plausible. RNAi has been successful in avian embryos and tissues (23,24) and porcine cell culture (25,26). However, there have been no reports of the generation of a transgenic livestock animal that exhibits RNAi. Literature reporting development of transgenic mice displaying stable germ-line transmittance of "genes" for gene-specific RNAi has been available for several years (27,28). The lentiviral-based system used to generate these transgenic gene-silencing mice has been successfully employed for generation of transgenic livestock (29,30), so the methodologies required for creating livestock transgenic for gene silencing are available. Recently, genetically modified pigs have been produced with a nonviral vector (31), which may curb concerns from the public regarding the safety of the consumption of products that come from genetically modified animals created using lentiviral methods. Additionally, the adeno-associated viral delivery of genetic information may allow avoidance of the phrase "genetically modified livestock," because this technique allows long-term expression of a transgene without integration into the host genome (32).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: B. M. Cleveland, A. S. Kiess, and K. P. Blemings, no conflicts of interest.
3 This is scientific article number 3011 of the West Virginia Agricultural and Forestry Experiment Station.
4 Present address: National Center for Cool and Cold Water Aquaculture, 11861 Leetown Rd., Kearneysville, WV 25430.
5 Present address: Mississippi State University, Poultry Science Department, Box 9665, Mississippi State, MS 39762.
6 Abbreviations used: ARP, acidic ribosomal protein; AASS, -aminoadipate -semialdehyde synthase; LKR, lysine -ketoglutarate reductase; LOX, lysine oxidation; RNAi, RNA interference; shRNA, short hairpin RNA.
Manuscript received 15 May 2008. Initial review completed 12 June 2008. Revision accepted 31 July 2008.
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LITERATURE CITED |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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