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Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

Cheng YF, Young GH, Lin JT, Jang HH, Chen CC, Nong JY, Chen PK, Kuo CY, Kao SH, Liang YJ, Chen HM - PLoS ONE (2015)

Bottom Line: Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC.Similarly, the expression of a shRNA against APRT ified the anti-inflammatory effects of adenine in HUVECs.These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

View Article: PubMed Central - PubMed

Affiliation: Energenesis Biomedical Co., Ltd., New Taipei City, Taiwan.

ABSTRACT
The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT ified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

No MeSH data available.


Related in: MedlinePlus

Effect of adenine induced AMPK activation and monocyte adhesion by APRT in HUVECs.(A) shNT HUVECs or shAPRT HUVECs were treated with various concentration of adenine for 6 h. Cell lysates collected from each condition were used to determine the phosphorylation of AMPK and ACC by western blot using antibodies specific for the phosphorylated protein. (B) shNT HUVECs or shAPRT HUVECs were exposed to 10 μg/L of TNF-α for 6 h in the presence or absence of 600 μM adenine. Top: representative images depicting monocytes to the HUVEC cells as described in “Material and methods”, Scale bar: 200 μM. Bottom: quantification of monocyte adhesion to HUVEC cells. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). ***, P<0.001; N.S., no significance.
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pone.0142283.g006: Effect of adenine induced AMPK activation and monocyte adhesion by APRT in HUVECs.(A) shNT HUVECs or shAPRT HUVECs were treated with various concentration of adenine for 6 h. Cell lysates collected from each condition were used to determine the phosphorylation of AMPK and ACC by western blot using antibodies specific for the phosphorylated protein. (B) shNT HUVECs or shAPRT HUVECs were exposed to 10 μg/L of TNF-α for 6 h in the presence or absence of 600 μM adenine. Top: representative images depicting monocytes to the HUVEC cells as described in “Material and methods”, Scale bar: 200 μM. Bottom: quantification of monocyte adhesion to HUVEC cells. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). ***, P<0.001; N.S., no significance.

Mentions: The utilization of adenine depends on the activity of APRT via the incorporation of PRPP into AMP [25]. To determine whether the effect of adenine-induced AMPK phosphorylation is mediated through the activity of APRT, we used retroviral short hairpin RNA (shRNA) against APRT in HUVECs. The expression of knockdown APRT was verified by western blot analysis and compared with the non-targeted shRNA (shNT) in HUVECs. We found that HUVECs transfected with a shAPRT plasmid exhibited a 92% reduction in APRT compared with the shNT HUVECs (S1B Fig). As shown in Fig 6A (Right panel), the levels of phosphorylated AMPK and its downstream target ACC increased in a dose-dependent manner following adenine treatment in shNT HUVECs, whereas they were unchanged in shAPRT HUVECs (Fig 6A, Left panel). This suggests that adenine-induced AMPK phosphorylation is mediated by the activity of APRT. Similarly, the effect of adenine on the adhesion between THP-1 cells and HUVECs was not changed by knockdown in HUVECs (Fig 6B). These results indicate that adenine mediates the TNF-α-induced adhesion ability between HUVECs and THP-1 cells via APRT.


Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

Cheng YF, Young GH, Lin JT, Jang HH, Chen CC, Nong JY, Chen PK, Kuo CY, Kao SH, Liang YJ, Chen HM - PLoS ONE (2015)

Effect of adenine induced AMPK activation and monocyte adhesion by APRT in HUVECs.(A) shNT HUVECs or shAPRT HUVECs were treated with various concentration of adenine for 6 h. Cell lysates collected from each condition were used to determine the phosphorylation of AMPK and ACC by western blot using antibodies specific for the phosphorylated protein. (B) shNT HUVECs or shAPRT HUVECs were exposed to 10 μg/L of TNF-α for 6 h in the presence or absence of 600 μM adenine. Top: representative images depicting monocytes to the HUVEC cells as described in “Material and methods”, Scale bar: 200 μM. Bottom: quantification of monocyte adhesion to HUVEC cells. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). ***, P<0.001; N.S., no significance.
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Related In: Results  -  Collection

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pone.0142283.g006: Effect of adenine induced AMPK activation and monocyte adhesion by APRT in HUVECs.(A) shNT HUVECs or shAPRT HUVECs were treated with various concentration of adenine for 6 h. Cell lysates collected from each condition were used to determine the phosphorylation of AMPK and ACC by western blot using antibodies specific for the phosphorylated protein. (B) shNT HUVECs or shAPRT HUVECs were exposed to 10 μg/L of TNF-α for 6 h in the presence or absence of 600 μM adenine. Top: representative images depicting monocytes to the HUVEC cells as described in “Material and methods”, Scale bar: 200 μM. Bottom: quantification of monocyte adhesion to HUVEC cells. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). ***, P<0.001; N.S., no significance.
Mentions: The utilization of adenine depends on the activity of APRT via the incorporation of PRPP into AMP [25]. To determine whether the effect of adenine-induced AMPK phosphorylation is mediated through the activity of APRT, we used retroviral short hairpin RNA (shRNA) against APRT in HUVECs. The expression of knockdown APRT was verified by western blot analysis and compared with the non-targeted shRNA (shNT) in HUVECs. We found that HUVECs transfected with a shAPRT plasmid exhibited a 92% reduction in APRT compared with the shNT HUVECs (S1B Fig). As shown in Fig 6A (Right panel), the levels of phosphorylated AMPK and its downstream target ACC increased in a dose-dependent manner following adenine treatment in shNT HUVECs, whereas they were unchanged in shAPRT HUVECs (Fig 6A, Left panel). This suggests that adenine-induced AMPK phosphorylation is mediated by the activity of APRT. Similarly, the effect of adenine on the adhesion between THP-1 cells and HUVECs was not changed by knockdown in HUVECs (Fig 6B). These results indicate that adenine mediates the TNF-α-induced adhesion ability between HUVECs and THP-1 cells via APRT.

Bottom Line: Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC.Similarly, the expression of a shRNA against APRT ified the anti-inflammatory effects of adenine in HUVECs.These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

View Article: PubMed Central - PubMed

Affiliation: Energenesis Biomedical Co., Ltd., New Taipei City, Taiwan.

ABSTRACT
The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT ified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

No MeSH data available.


Related in: MedlinePlus