Stable over-expression of the human malate–aspartate NADH shuttle member Aralar I in PK15 cells improves energy metabolism and enhances proliferation of porcine circovirus-2

Abstract

The considerable losses sustained by the pig industry due to porcine circovirus-2 (PCV2) could be avoided by using an attenuated vaccine. However, production of an attenuated PCV2 vaccine has not been satisfactory. Recently, we over-expressed the human malate–aspartate NADH shuttle member Aralar I in PK15 cells to enhance proliferation of PCV2. Compared with control PK15 cells, lactate accumulation in Aralar I over-expressing cells decreased by 44.8% (p < 0.01), whereas the mitochondrial NADH and pyruvate concentrations increased by 50% (p < 0.05) and 35.4% (p < 0.05), respectively. The ATP/ADP ratio and O₂ uptake rate also increased by 39% (p < 0.05) and 37.8% (p < 0.01), respectively. Furthermore, the glutamine consumption rate increased by 36.4% (p < 0.01). Finally, compared with the PCV2 yield of infected control PK15 cells, the PCV2 yield of infected Aralar I over-expression cells was enhanced by 42.5% (p < 0.05). These results revealed that over-expression of Aralar I can improve the energy metabolism of PK15 cells and also enhance the replication of the PCV2 virus in PK15 cells. This study presents a method to improve energy metabolism and provides a valuable thinking to enhance virus vaccine production.

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1. Introduction

PCV2 is the causative agent of several piglet diseases and syndromes including porcine dermatitis and nephropathy syndrome (PDNS), late-term abortions, reproductive failure in sows, proliferative and necrotizing pneumonia (PNP) and congenital defects.^1–5 Among these conditions, postweaning multisystemic wasting syndrome (PMWS) is the most important.⁶ PCV2 has led to considerable losses for the global pig industry. Approximately 33–40% of newborn and pre-suckle piglets are PCV2-viremic.^7,8 PCV2 virus particles are very stable and are able to persist in the environment of infected herds, making virus eradication very difficult.⁹ Alternative strategies should therefore be investigated for disease control, and immune prophylaxis could be an adequate strategy. Currently, there are several types of PCV2 vaccines, including an inactive vaccine, attenuated vaccine, subunit vaccine and genetically engineered vaccine.^10–12

In mammalian cells, glucose is oxidized to pyruvate and generates NADH through the glycolytic pathway. Pyruvate is vital to energy metabolism. In mammalian cells, there are two major pyruvate metabolism pathways. Pyruvate dehydrogenase can catalyze pyruvate to acetyl Co-A, which then enters the TCA cycle and is oxidized to NADH. Finally, NADH is oxidized and generates ATP to provide energy for cellular metabolism. Pyruvate can also be catalyzed by lactate dehydrogenase. In this pathway, lactate is produced. Previous research has indicated that mammalian cells cultured in vitro consume a large amount of glucose, of which approximately 90% is catalyzed to lactate.^13,14 Why mammalian cells cultured in vitro consume so much glucose to generate lactate is still unclear. One hypothesis is that the NADH shuttle systems, such as the malate–aspartate NADH shuttle, in mammalian cells are not very efficient, and the NADH that accumulates in the cytoplasm is consumed by lactate dehydrogenase, which is needed to catalyze pyruvate to lactate.¹⁵ Thus the cells exhibited a less efficient energy metabolism, this decrease in energy efficiency may be due to an inability of pyruvate to progress into the TCA cycle and NADH shuttle systems. The lack of progression into the TCA cycle or overflow metabolism resulted in the inadequate supply of ATP for the cells.¹⁶

NADH and its oxidized form, NAD⁺, are the most important coenzymes found in all living cells. The NAD⁺/NADH redox state is a central metabolic node.¹⁷ These coenzymes participate and play critical roles in multiple biological processes, including energy metabolism, mitochondrial function, calcium homeostasis, biosynthesis, cell death and gene expression.^18–23 The most important function of NADH is being oxidized to generate ATP, which provides energy for almost all cellular metabolism. In mammalian cells, NADH is generated from glycolysis and the TCA cycle. NADH from the cytoplasmic glycolysis must be transported to the mitochondria to be oxidized. The NADH shuttle must transport NADH into the mitochondria because NADH cannot be transported into the mitochondria by itself. Previous research has indicated that glycolytic flux, pyruvate metabolism and lactate metabolism are all affected by the NADH shuttle. Glycolytic flux is constrained by how rapidly NADH can be regenerated to NAD⁺ through conversion of pyruvate to lactate and by the NADH shuttle. At steady state, the rate of pyruvate entering the tricarboxylic acid (TCA) cycle is approximately the same as the flux of reducing equivalents transferred across the mitochondrial membrane by the NADH shuttle. To divert more pyruvate into the mitochondria, NADH transfer into the mitochondria must also be increased. The rate of lactate transport into the cell is constrained by the fluxes of pyruvate transport and the NADH shuttle into the mitochondria. Increasing the activity of NADH shuttles in CHO and NS0 cells has led to increased transport of pyruvate into the mitochondria, thus either decreasing lactate production or increasing its consumption.²⁴

Aralar I is a critical carrier protein in the malate–aspartate NADH shuttle system. It belongs to the mitochondria transporter family SLC25.²⁵ The expression of Aralar I in tissue is specific. It is primarily expressed in excitable tissues, including brain, heart and skeletal muscle. The expression levels are very low in kidney, liver and lung.²⁶ The expression level of Aralar I affects cell metabolism significantly, manifesting as over-expression of Aralar I in β-cells and increases in NADH concentration, mitochondrial membrane potential, ATP concentration and glutamine concentration, though lactate accumulation decreases.²⁷

A virus is a simple life form that cannot grow and proliferate independently. The energy needed to proliferate depends entirely on the energy metabolism of the host cells. Thus, the proliferation of a virus is significantly affected by the energy state of host cells.²⁸ We suppose that improving the energy state of host cells by enhancing the flux of NADH and pyruvate transported into the mitochondria, the proliferation of virus in host cells will also be enhanced. Indeed, the results confirmed our supposition. Here, we over-expressed the Aralar I protein in PK15 cells, and we detected changes in the cell growth, glucose metabolism, lactate metabolism, NADH metabolism, pyruvate metabolism, ATP/ADP ratio, O₂ uptake rate, glutamine metabolism and PCV2 proliferation of PK15 cells at each stage of cell growth. The results indicated that the over-expression of Aralar I in PK15 cells, flux of NADH and pyruvate transport into the mitochondria were all enhanced. The ATP/ADP ratio, O₂ uptake rate and glutamine consumption all increased. The accumulation of lactate decreased significantly and the proliferation of PCV2 in PK15 cells was indeed enhanced. This study provided a new way to improve virus vaccines production.

2. Materials and methods

No violation of human or animal rights.

2.1. Construction of Aralar I over-expressed cell clones

Nhe I and Not I are digestion sites at the 5′ and 3′ ends of the human aralar I gene (NCBI number NM-003705) in the pUC19 vector, respectively. After digestion at Nhe I and Not I, the aralar 1 gene was constructed into the pCI vector. PK15 cells were maintained in modified Eagle's medium (MEM, Gibco) with 5% FBS (fetal bovine serum, Gibco). The culture condition was 37 °C in a humidified atmosphere of 5% CO₂. Before transfection, 2 ml of the PK15 cells at a concentration of 2.5 × 10⁵ cells per ml were seeded into each well of a 6-well plate. After incubating for 16 hours, 2.5 μg pCI-aralar 1 DNA and lacZ DNA were transfected into the PK15 cells mediated by Lipofectamine 2000 (Invitrogen), respectively. Twenty-four hours later, the medium was changed to MEM with 5% FBS and 800 μg ml⁻¹ G418 (Sigma). The medium was replaced with fresh medium every 48 hour until cell growth was established, and then, cell clones were screened by FACS. PK15 cells expressing lacZ were set as control.

2.2. mRNA level and western blot analysis

1 ml TRIzol reagent (Invitrogen) was added into 5.0 × 10⁶ cells. After incubate for 5 minute at 25 °C, 0.2 μl chloroform was added and oscillated for 5 second. After centrifuging 15 minute at 12 000 rpm and 4 °C, 500 μl supernatant was transferred into an RNase free EP tube, and 500 μl isopropanol was also added into it. Incubation for 20 minute followed by centrifuging 10 minute at 12 000 rpm. The sediment, total RNA, was washed twice with 75% alcohol and dissolved with 20 μl RNase free water. Then RNA was taken for cDNA synthesis. RNA PCR Kit (TaKaRa Dalian Biotechnology Co., Ltd. Dalian, China) was used for reverse transcription PCR according to the manufacturer's instruction. Reverse transcription was performed in a final volume of 10 ml, containing 0.5 μl Oligdt, 2 μl MgCl₂ (2.5 mM), 1 μl 10× RNA PCR buffer, 1 μl dNTP mixture, 0.5 μl reverse transcriptase, 4 μl RNA and 1 μl DEPC-treated H₂O. RT was performed with the following program: 10 min at 30 °C, 30 min at 42 °C, 5 min at 99 °C and 5 min at 4 °C. PCR was performed in a total reaction volume of 50 μl reaction mixture by adding 40 μl of mixture, containing 10 μl of 5 × PCR buffer, 28.75 μl of sterilized distilled water, 0.25 μl of Taq and 0.5 μl each primer (for Aralar I, forward primer was 5′-ata GCTAGC atggcggtcaaggtgcag acaac-3′ and the reverse primer was 5′-ata GCGGCCGC tcactgagtggctgccactg-3′). PCR was performed in a cycling condition as following: 2 min at 94 °C followed by 30 cycles of 30 s at 94 °C, 1 min at 55 °C and 2 min at 72 °C with a final step at 72 °C for 3 min to allow complete extension of all amplified fragments. The amplified products of Aralar I had an expected size of 2037 bp on 1% agarose gel electrophoresis.

The cell protein extracts (50 μg) of control PK15 cells and Aralar I over-expressed cells were prepared by detecting their total protein concentration. Samples were subsequently subjected to SDS/PAGE on 7.5% (w/v) polyacrylamide gels. Proteins were transferred onto a nitrocellulose membrane. After blocking in 5% (v/v) milk protein, membranes were then probed with anti-Aralar I antibodies (AVIVA Systems Biology; diluted to 1 : 1000), washed three times with PBS and detected with HRP-labeled goat anti-rabbit IgG (ProteinTech; diluted to 1 : 2000). β-Actin was used as a control.

2.3. Measurement of cell numbers and concentrations of glucose, lactate, glutamine

We injected 25 μl of the control PK15 cells or Aralar I over-expression cell culture supernatant into a biochemical analyzer (Shandong Institute of Life Science) to detect the concentration of glucose and lactate. We added 700 μl control PK15 cells or Aralar I over-expression cell culture supernatant to an EP tube, and 60 μl trichloroacetic acid (50%) were also added. We centrifuged for 10 min at 12 000 rpm, and then, the supernatant was filtered using a nitrocellulose membrane. Next, 200 μl filter liquor was used to detect the concentration of glutamine with HPLC. Cells were digested and resuspended, and then dyed with trypan blue and counted in a blood counting chamber.

2.4. Mitochondria extraction

Cultured control cells or Aralar I over-expressed cells were digested and resuspended in cold PBS. The cell density was diluted to 1.0 × 10⁶ cells per ml, and 2 ml cell supernatant was added to a 10 ml tube and the tube was placed on ice. Cells were broken with an ultrasonic instrument (ATPIO) at 10% power. The program was turned on for 3 s and turned off for 9.9 s for 3 cycles. Then, we centrifuged for 10 min at 600 rpm. Mitochondria were extracted from the supernatant using a mitochondria extraction kit (Biovision).

2.5. NADH measurement

Control cells or Aralar I over-expressed cells (1.0 × 10⁶ cells) were used to detect the total NADH concentration or mitochondrial NADH concentration following mitochondria extraction. NADH was detected with a NADH Assay Kit (Bioassay Systems). Briefly, 40 μl NADH extraction buffer was added to the cell or mitochondria sample and then incubated for 5 min in 60 °C water. Next, 8 μl assay buffer and 40 μl NAD extraction buffer were added, and the samples were centrifuged for 5 min at 4 °C and 14 000 rpm. Then, we added 60 μl work reagent to the 40 μl supernatant to detect the NADH concentration of the samples.

2.6. Pyruvate measurement

We used 1.0 × 10⁶ cells of the Control PK15 cells or Aralar I over-expressed cells to detect total pyruvate concentration or mitochondria pyruvate concentration following mitochondria extraction. Pyruvate was detected with a pyruvate assay kit (Nanjing Jiancheng Bioengineering Institute). Cells or mitochondria were resuspended with 100 μl PBS. And then, samples were frozen at −80 °C for 10 min followed by 25 °C for 20 min and repeated three times. Next, we added work reagents and detected the concentration.

2.7. ATP/ADP ratio assay

The ATP/ADP ratio was detected with an ATP/ADP ratio assay kit (Bioassay Systems). Control cells or Aralar I over-expressed cells (1.0 × 10⁶ cells) were diluted with 100 μl deionized water. Then, the 10 μl suspension was added into each well of a white opaque 96-well plate. For each well, 60 μl ATP reagent was added. Luminescence was detected 1 min later and detected again after 10 min. Next, 5 μl ADP reagent was added and luminescence was detected immediately.

2.8. O₂ consumption rate detection

The O₂ consumption rate was detected with a respirometer (OROBOROS Oxygraph 2K). The cell density of the control cells or Aralar I over-expressed cells was diluted to 1.0 × 10⁶ cells per ml. We added 2.1 ml fresh medium to each chamber of the Oxygraph 2K to calibrate the saturation oxygen concentration, and then, we replaced the medium with fresh medium including cells. When the curves representing the O₂ consumption rate were stable, we calculated the O₂ consumption rate.

2.9. PCV2 infection and PCV2 titer measurement

Control cells or Aralar I over-expressed cells were seeded into T25 cell culture flasks at 2.5 × 10⁵ cells per ml × 5 ml per flask, and we added an extra 5 ml medium with 6 mM D-glucosamine and 5% (v/v) PCV2 (TCID₅₀ = 10^−4.6/0.1 ml). Cells were incubated at 37 °C for 48 hour. Media were replaced by 10 ml MEM with 3% (v/v) FBS and 3 mM D-glucosamine and cultured for another 48 hour. Before measuring the titer, the control PK15 cells and Aralar 1 over-expressed cells had to be broken by repeated freezing and thawing and serially diluted to 1 : 10 with MEM containing 6 mM D-glucosamine. For each sample, six dilutions, 10⁻¹ to 10⁻⁶, were used to measure the infection ratio. Each dilution was seeded in six wells, with 100 μl in each well. We simultaneously added a 100 μl cell suspension into each well. Blank controls were needed. The plates were incubated for 72 hours at 37 °C with 5% CO₂. Then, the suspensions were discarded, and each well was washed with 200 μl PBS. The plates were dried, and 100 μl cold ethanol was added to each well. The ethanol was discarded, and the dried plates were anchored at −20 °C. The plates were washed again with PBS, and 100 μl 1 : 400 diluted anti PCV2 serum (KPL) was added to each well. The plates were incubated for 1 h at 37 °C, followed by three washes with PBS. SPA (KPL) diluted to 1 : 3000 was added at 50 μl per well, and 30 min later, each well was washed with distilled water, and the infected wells were counted. TCID50 was determined by the Reed–Muench method.

2.10. Statistical analysis

Data are the mean ± S.E. for at least three independent experiments performed in triplicate. Differences between groups were assessed by the Student's t test for unpaired data.

3. Results

3.1. Over-expression of Aralar I in PK15 cells

We obtained three cell clones overexpressing Aralar I, called A1, A21 and A22. The mRNA and protein expression of Aralar I in the control cells and three clone cells were detected. Aralar I was over-expressed in the A1, A21 and A22 cell clones in mRNA levels (Fig. 1A), and was not expressed in control cells. Compared with the protein expression of Aralar I in control cells, Aralar I was over-expressed in the A1, A21 and A22 cell clones (Fig. 1B). Of these clones, the A21 clone had the highest expression level of Aralar I.

Fig. 1 Expression of Aralar 1 and β-actin in control cells and A1, A21, A22 clone cells. (A) mRNA level expression analysis of human Aralar I gene.“−” was β-actin cDNA which was 1127 bp.“+” was human Aralar I cDNA which was 2037 bp. (B) Protein was extracted and concentration was measured. 50 μg total protein were used to detect Aralar I. Bands correspond to 70 kDa were Aralar I. At 45 kDa, bands correspond to β-actin.

3.2. Effect of Aralar I over-expression on cell growth

The cell density of the control cells or A21 cells was measured every 24 hours. The results showed that in the whole growth stage, there were no significant differences in the cell densities of control cells and A21 cells (Fig. 2). After 96 hours of cell growth, the cell density of control cells was 1.98 × 10⁶ cells per ml (p < 0.05), whereas the cell density of A21 cells was 2.1 × 10⁶ cells per ml (p < 0.05). The results showed that over-expression of Aralar I has no obvious effect on the growth of PK15 cells.

Fig. 2 Effect of Aralar 1 over-expression on cell growth. Cell densities of control cells or A21 cells were measured every 24 h. Values are means ± S.D. of three independent experiments performed in quadruplicate p < 0.05.

3.3. Effect of Aralar 1 over-expression on glucose consumption and lactate production

The supernatant of cultured control cells or A21 cells was used to measure glucose or lactate concentration every 24 hours. We found that throughout all stages of cell growth, there were no obvious differences in the glucose consumption rates of control cells and A21 cells (Fig. 3A), however there were significant differences in lactate production (Fig. 3B) and the yield of lactate to glucose (Fig. 3C). The differences in lactate production and yield of lactate to glucose were the highest at 48 hour in A21 cells compared with control cells. The lactate accumulation and the yield of lactate to glucose in A21 cells decreased by 44.8% (p < 0.01) and 41.9% (p < 0.01), respectively. These results indicated that over-expression of Aralar I could reduce the production of lactate and increase the utilization efficiency of glucose.

Fig. 3 Effect of Aralar I over-expression on glucose consumption and lactate production. (A) Glucose concentration of control cells or A21 cells were measured every 24 h. (B) Lactate concentration of control cells or A21 cells were measured every 24 h. (C) Yield of lactate to glucose. Values were means ± S.D. of three independent experiments performed in quadruplicate p < 0.01 compared with control cells at corresponding conditions.

3.4. Effect of Aralar I over-expression on intracellular NADH metabolism

We researched the effect of Aralar I over-expression on total NADH and mitochondrial NADH. The total NADH concentration in A21 cells was higher than in control cells throughout all cell growth stages (Fig. 4A). At 48 hour of cell growth, the difference in the total NADH concentration in control cells and A21 cells was the most significant. At 48 hour, compared with control cells, the total NADH concentration in A21 cells was increased by 32.5% (p < 0.05).

Fig. 4 Effect of Aralar I over-expression on intracellular NADH metabolism. 1.0 × 10⁶ cells of control cells or A21 cells were used to detect total NADH concentration (A) or mitochondria NADH concentration (B) following mitochondria extraction every 24 h. Values are means ± S.D. of three independent experiments performed in quadruplicate p < 0.05 compared with control cells at corresponding conditions.

The mitochondrial NADH concentration in A21 cells was also higher than in control cells throughout all cell growth stages (Fig. 4B). The maximum difference in the mitochondrial NADH concentration of control cells and A21 cells was also at 48 hour of cell growth. At 48 hour, the mitochondrial NADH concentration in A21 cells increased by 50% (p < 0.05) compared with that of control cells. Aralar I over-expression could enhance intracellular NADH metabolism while improving the flux of NADH transferring into the mitochondria.

3.5. Effect of Aralar I over-expression on intracellular pyruvate metabolism

The total pyruvate concentration in A21 cells was higher than in control cells throughout all cell growth stages (Fig. 5A). At 48 hour of cell growth, the difference in the total pyruvate concentration of control cells and A21 cells was the most significant. At 48 hour, compared with the total pyruvate concentration of control cells, the total pyruvate concentration of A21 cells was increased by 33.8% (p < 0.05).

Fig. 5 Effect of Aralar I over-expression on intracellular pyruvate metabolism. 1.0 × 10⁶ cells of control cells or A21 cells were used to detect total pyruvate concentration (A) or mitochondria pyruvate concentration (B) following mitochondria extraction every 24 h. Values were means ± S.D. of three independent experiments performed in quadruplicate p < 0.05 compared with control cells at corresponding conditions.

The mitochondrial pyruvate concentration in A21 cells was also higher than in control cells throughout all cell growth stages (Fig. 5B). The maximum difference between the mitochondrial pyruvate concentration of control cells and A21 cells was also at 48 hour of cell growth. At 48 hour, the mitochondrial pyruvate concentration of A21 cells was 35.4% (p < 0.05) higher than that of control cells. These results indicated that Aralar I over-expression can enhance the metabolism of total pyruvate and also improve the flux of pyruvate transferring into the mitochondria.

3.6. Effect of Aralar I over-expression on ATP/ADP ratio and O₂ consumption rate

Compared with control cells, the O₂ consumption of A21 cells was higher in every stage of cell growth (Fig. 6A). Interestingly, the difference was also particularly obvious at 48 hour of cell growth. At this time, compared with control cells, the O₂ consumption of A21 cells was increased by 37.8. The effect of Aralar 1 over-expression on ATP/ADP ratio was almost the same as the O₂ consumption (Fig. 6B). At 48 hour, the O₂ consumption rate of A21 cells was enhanced by 39% (p < 0.01) compared with that of control cells. These results indicated that over-expressing Aralar I could enhance the O₂ consumption rate and ATP/ADP ratio, which means that the energy metabolism of PK15 cells was improved.

Fig. 6 Effect of Aralar I over-expression on ATP/ADP and O₂ consumption rate. For measuring the O₂ consumption rate (A), cells were resuspended and diluted with cell cultured supernatant. When measuring the ATP/ADP ratio (B), cells were diluted with 100 μl distill water and measured immediately. Values are means ± S.D. of three independent experiments performed in quadruplicate p < 0.05 compared with control cells at corresponding conditions.

3.7. Effect of Aralar I over-expression on glutamine

The supernatant of the cultured control cells or A21 cells was used to measure the glutamine concentration every 24 hour. We found that in every stage of cell growth, the glutamine consumption rate of A21 cells was higher than that of control cells (Fig. 7). The maximum differentiation was also at 48 hour. At this time, the glutamine consumption rate of A21 cells was enhanced by 36.4% (p < 0.01). These results indicated that over-expressing Aralar 1 could also enhance the glutamine metabolism of PK15 cells.

Fig. 7 Effect of Aralar 1 overexpression on glutamine metabolism. Glutamine concentration were measured with HPLC. Values are means ± S.D. of three independent experiments performed in quadruplicate p < 0.01 compared with control cells at corresponding conditions.

3.8. The metabolism differences between PCV2 infected control cells and PCV2 infected A21 cells

When infected by PCV2, the glucose consumption rate of A21 cells and control cells had no obvious difference at 48 hour (Fig. 8A). But the lactate production rate of A21 cells and control cells had significant difference. The difference between A21 cells and control cells in lactate production rate was up to 96.3% (p < 0.01) (Fig. 8B). The lactate production rate of A21 cells decreased significantly. Compared with PCV2 infected control cells, the total NADH concentration, mitochondrial NADH concentration (Fig. 8C), total pyruvate concentration, mitochondrial pyruvate concentration (Fig. 8D), ATP/ADP ratio (Fig. 8F) in PCV2 infected A21 cells were all increased, but the difference decreased. The O₂ (Fig. 8E) and glutamine (Fig. 8G) consumption rate of PCV2 infected A21 cells and the difference with PCV2 infected control cells were all increased. This may be due to Aralar I over-expression cells to produce more PCV2 and need to consume more metabolites. These results indicated that being infected by PCV2, host cells need extra energy for virus replication.

Fig. 8 Effect of Aralar 1 over-expression on 48 h metabolisms of PCV2 infected cells. After 48 hours of growth, glucose consumption rate (A), lactate production rate (B), NADH concentration (C), pyruvate concentration (D), O₂ consumption rate (E), ATP/ADP value (F) and glutamine consumption rate (G) of PCV2 infected control cells and PCV2 infected A21 cells were detected respectively. Values were means ± S.D. of three independent experiments performed in quadruplicate p < 0.05.

3.9. Effect of Aralar I over-expression on PCV2 proliferation in PK15 cells

Control cells and A21 cells were infected by PCV2, and the TCID₅₀ was measured every 24 hours. After 24 hours of infection, there were no significant differences between the TCID₅₀ of control cells and A21 cells (Fig. 9). The difference in the TCID₅₀ of control cells and A21 cells was obvious from 48 hour to 96 hour. Finally, after infection for 96 hour, the PCV2 titer, which was detected in infected A21 cells, increased by 42.5% (p < 0.05) compared with that of control cells. These results indicated that over-expressing Aralar I in PK15 cells could enhance PCV2 proliferation in PK15 cells.

Fig. 9 Effect of Aralar I over-expression on PCV2 proliferation in PK15 cells. Before measuring the TCID₅₀, cells would be broken by freezing and thawing. Values were means ± S.D. of three independent experiments performed in quadruplicate p < 0.05 compared with control cells at corresponding conditions.

4. Discussion

PCV2 attenuated vaccine is a PCV2 virus which have no strong pathogenic. It is one of useful strategies to protect pigs from diseases which caused by PCV2 infection.^29–32 But the titer of this kind of PCV2 virus is not satisfactory in industry. In our study, we over-expressed Aralar I to enhance energy metabolism in host cells and as a result, the titer of this kind of PCV2 also enhanced.

Lactate accumulates as the end product of glycolysis when oxidative phosphorylation, as well as the tricarboxylic acid cycle are reduced.^33–36 A previous study indicated that the accumulation of lactate is constrained by the fluxes of the NADH shuttle into the mitochondria. Increasing the activity of NADH shuttles in cells could possibly lead to increased transport of pyruvate into the mitochondria, thus either decreasing lactate production or increasing its consumption.²⁴ In our present study, the host PK15 cells indicates a very low glucose utilization efficiency. Along with cell growth, the glucose in PK15 cells was consumed, and a large amount of lactate was simultaneously produced. However, the accumulation of lactate decreased significantly after over-expression of Aralar I. This may be because Aralar I over-expression increased the activity of NADH shuttles in cells which at last led to either decreasing lactate production or increasing its consumption. Aralar I is the critical protein in the malate–aspartate NADH shuttle.²⁶ A previous study indicated that Aralar I over-expression could increase total cell NADH concentration.³⁷ But it is still unclear how Aralar I over-expression affects mitochondrial NADH concentration. Here, we over-expressed Aralar 1 in PK15 cells. Then, we found that with Aralar I over-expressed in PK15 cells, the total NADH and mitochondrial NADH concentration both increased. This may be because over-expression of Aralar I can lead to more NADH being transported into the mitochondria, thus increasing the mitochondrial NADH concentration and simultaneously reducing lactate production and decreasing total NADH consumption. Over-expression of Aralar I could enhance glucose utilization efficiency by oxidizing the glucose to produce energy that may be oxidized to produce byproduct lactate in control cells. In this study, the mitochondrial NADH concentration detected was low. This may be because NADH was lost during the sample treatments, and the process of extracting the mitochondria may not have been sufficiently efficient. Currently, extracting mitochondria efficiently and exactly detecting the mitochondrial NADH concentrations of adherent cells are still very difficult.

Aralar I over-expression can increase intracellular ATP/ADP ratio significantly, especially at 48 hour. This may be because at 48 hour, cells are experiencing exponential growth and cell metabolisms are more active. More ATP is needed to support nuclear replication and protein expression. In control cells, more glucose would be shifted toward the production of lactate instead of toward TCA. After Aralar I was over-expressed, glucose utilization and energy metabolism efficiency could be improved.

Previous research has reported that over-expression of Aralar I in CHO cells can enhance the transportation of pyruvate into the mitochondria.²⁶ Here, we obtained the same results with PK15 cells. Over-expression of Aralar I in PK15 cells significantly increased total pyruvate, especially mitochondrial pyruvate. We inferred that Aralar I over-expression can not only enhance the transportation of pyruvate into mitochondria, but also enhance the metabolism of pyruvate production.

Glutamine is very important to mammalian cells cultured in vitro.³⁸ The consumption rate of glutamine has a relationship with the activity of TCA cycle.³⁹ We found that over-expression of Aralar I can also enhance the consumption rate of glutamine. This may be caused by an enhanced TCA cycle. With over-expression of Aralar 1 in PK15 cells, more pyruvate was transported into the mitochondria and oxidized through the TCA cycle. The enhanced TCA cycle needs more α-oxoglutarate, which is generated from glutamine metabolism.

The proliferation of virus depends entirely on the energy state of host cells.²⁸ In the PCV2 propagation process in this research, the effects of Aralar I over-expression on cellular glucose, lactate, NADH, pyruvate and glutamine metabolism were similar to the cell growth process. But the metabolic differences between A21 cells and control cells were reduced, especially at 48 hour (Fig. 8). This may be because Aralar I over-expression cells need more ATP to produce more PCV2. The enhancement of the O₂ consumption rate indicated that the metabolism of ATP was still enhanced. Aralar I over-expression enhanced cellular energy metabolism, as a result, the proliferation of PCV2 also enhanced.

5. Conclusion

To summarize, by improving the energy metabolism of PK15 cells to enhance PCV2 proliferation was investigated for the first time. After over-expression of Aralar I, the accumulation of lactate decreased, and glucose utilization efficiency was enhanced in every stage of cell growth. Compared with control cells, PK15 cells over-expressing Aralar I had a high mitochondrial NADH concentration, mitochondrial pyruvate concentration, ATP/ADP ratio, O₂ consumption rate and glutamine consumption rate. In general, PK15 cells over-expressing Aralar I had enhanced energy metabolism, which enhanced the proliferation of PCV2.

Conflict of interest

No conflict of interest.

Acknowledgements

This work was supported by grants from the Shanghai Zhangjiang National Innovation Demonstration Zone Key projects of Special Development Funds (201310-FX-B2-002) and the Chinese National Programs for High Technology Research and Development (2015AA020801).

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Footnote

† These authors contributed equally.

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