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Glutamine starvation enhances PCV2 replication via the phosphorylation of p38 MAPK, as promoted by reducing glutathione levels.

Chen X, Shi X, Gan F, Huang D, Huang K - Vet. Res. (2015)

Bottom Line: The results show that glutamine promoted PK-15 cell viability.Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells.Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. cxx@njau.edu.cn.

ABSTRACT
Glutamine has a positive effect on ameliorating reproductive failure caused by porcine circovirus type 2 (PCV2). However, the mechanism by which glutamine affects PCV2 replication remains unclear. This study was conducted to investigate the effects of glutamine on PCV2 replication and its underlying mechanisms in vitro. The results show that glutamine promoted PK-15 cell viability. Surprisingly, glutamine starvation significantly increased PCV2 replication. The promotion of PCV2 replication by glutamine starvation disappeared after fresh media with 4 mM glutamine was added. Likewise, promotion of PCV2 was observed after adding buthionine sulfoximine (BSO). Glutamine starvation or BSO treatment increased the level of p38 MAPK phosphorylation and PCV2 replication in PK-15 cells. Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells. Promotion of PCV2 replication caused by glutamine starvation could be blocked in p38-knockdown PK-15 cells. Therefore, glutamine starvation increased PCV2 replication by promoting p38 MAPK activation, which was associated with the down regulation of intracellular glutathione levels. Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.

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Effects of glutamine on viability of PK-15 cells. PK-15 cells without (A) or with (B) PCV2 infection were grown in 96-well plates with DMEM containing 4 mM glutamine for 24 h until 50% confluence was reached. Subsequently, the medium was changed to fresh DMEM with various concentrations of glutamine (0–16 mM). The cells were incubated for an additional 48 h before the cell viability was determined by MTT assay. Values are given as mean ± SD from three independent experiments. Groups were compared by a 1-way ANOVA followed by a least-significant difference test (*P < 0.05, **P < 0.01).
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Fig1: Effects of glutamine on viability of PK-15 cells. PK-15 cells without (A) or with (B) PCV2 infection were grown in 96-well plates with DMEM containing 4 mM glutamine for 24 h until 50% confluence was reached. Subsequently, the medium was changed to fresh DMEM with various concentrations of glutamine (0–16 mM). The cells were incubated for an additional 48 h before the cell viability was determined by MTT assay. Values are given as mean ± SD from three independent experiments. Groups were compared by a 1-way ANOVA followed by a least-significant difference test (*P < 0.05, **P < 0.01).

Mentions: To assess the effect of glutamine on the growth of PK-15 cells with or without PCV2 infection, PK-15 cells were grown in 96-well plates with DMEM containing 4 mM glutamine for 24 h until 50% confluence was reached. Subsequently, the medium was changed to fresh DMEM with various concentrations of glutamine (0–16 mM). The cells were incubated for an additional 48 h before the cell viability was determined. As shown in Figures 1A and 1B, the significant promotion of cell viability was observed when glutamine was present at 4 mM or higher as compared with groups subjected to glutamine starvation (P < 0.05). The cell viability was significantly decreased by 24%, 50%, 81%, and 87% in non-infected groups incubated with glutamine at concentrations of 1, 0.5, 0.1, and 0 mM, respectively, as compared with the controls with 4 mM glutamine treatment. The relative cell viability of the infected cells was 93%, 68%, 49%, 28%, and 23% when glutamine was supplemented at concentrations of 2, 1, 0.5, 0.1, and 0 mM, respectively. The promotion of PK-15 cell viability by glutamine was concentration dependent within the range of 0–2 mM glutamine. Our data indicate that glutamine plays an important role in PK-15 cell viability. Glutamine starvation inhibited the growth of PK-15 cells when its concentration was less than 4 mM in the medium.Figure 1


Glutamine starvation enhances PCV2 replication via the phosphorylation of p38 MAPK, as promoted by reducing glutathione levels.

Chen X, Shi X, Gan F, Huang D, Huang K - Vet. Res. (2015)

Effects of glutamine on viability of PK-15 cells. PK-15 cells without (A) or with (B) PCV2 infection were grown in 96-well plates with DMEM containing 4 mM glutamine for 24 h until 50% confluence was reached. Subsequently, the medium was changed to fresh DMEM with various concentrations of glutamine (0–16 mM). The cells were incubated for an additional 48 h before the cell viability was determined by MTT assay. Values are given as mean ± SD from three independent experiments. Groups were compared by a 1-way ANOVA followed by a least-significant difference test (*P < 0.05, **P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4363047&req=5

Fig1: Effects of glutamine on viability of PK-15 cells. PK-15 cells without (A) or with (B) PCV2 infection were grown in 96-well plates with DMEM containing 4 mM glutamine for 24 h until 50% confluence was reached. Subsequently, the medium was changed to fresh DMEM with various concentrations of glutamine (0–16 mM). The cells were incubated for an additional 48 h before the cell viability was determined by MTT assay. Values are given as mean ± SD from three independent experiments. Groups were compared by a 1-way ANOVA followed by a least-significant difference test (*P < 0.05, **P < 0.01).
Mentions: To assess the effect of glutamine on the growth of PK-15 cells with or without PCV2 infection, PK-15 cells were grown in 96-well plates with DMEM containing 4 mM glutamine for 24 h until 50% confluence was reached. Subsequently, the medium was changed to fresh DMEM with various concentrations of glutamine (0–16 mM). The cells were incubated for an additional 48 h before the cell viability was determined. As shown in Figures 1A and 1B, the significant promotion of cell viability was observed when glutamine was present at 4 mM or higher as compared with groups subjected to glutamine starvation (P < 0.05). The cell viability was significantly decreased by 24%, 50%, 81%, and 87% in non-infected groups incubated with glutamine at concentrations of 1, 0.5, 0.1, and 0 mM, respectively, as compared with the controls with 4 mM glutamine treatment. The relative cell viability of the infected cells was 93%, 68%, 49%, 28%, and 23% when glutamine was supplemented at concentrations of 2, 1, 0.5, 0.1, and 0 mM, respectively. The promotion of PK-15 cell viability by glutamine was concentration dependent within the range of 0–2 mM glutamine. Our data indicate that glutamine plays an important role in PK-15 cell viability. Glutamine starvation inhibited the growth of PK-15 cells when its concentration was less than 4 mM in the medium.Figure 1

Bottom Line: The results show that glutamine promoted PK-15 cell viability.Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells.Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. cxx@njau.edu.cn.

ABSTRACT
Glutamine has a positive effect on ameliorating reproductive failure caused by porcine circovirus type 2 (PCV2). However, the mechanism by which glutamine affects PCV2 replication remains unclear. This study was conducted to investigate the effects of glutamine on PCV2 replication and its underlying mechanisms in vitro. The results show that glutamine promoted PK-15 cell viability. Surprisingly, glutamine starvation significantly increased PCV2 replication. The promotion of PCV2 replication by glutamine starvation disappeared after fresh media with 4 mM glutamine was added. Likewise, promotion of PCV2 was observed after adding buthionine sulfoximine (BSO). Glutamine starvation or BSO treatment increased the level of p38 MAPK phosphorylation and PCV2 replication in PK-15 cells. Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells. Promotion of PCV2 replication caused by glutamine starvation could be blocked in p38-knockdown PK-15 cells. Therefore, glutamine starvation increased PCV2 replication by promoting p38 MAPK activation, which was associated with the down regulation of intracellular glutathione levels. Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases.

Show MeSH
Related in: MedlinePlus