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In vivo activation of AMP-activated protein kinase attenuates diabetes-enhanced degradation of GTP cyclohydrolase I.

Wang S, Xu J, Song P, Viollet B, Zou MH - Diabetes (2009)

Bottom Line: The administration of AICAR, an AMPK activator, or adenoviral overexpression of a constitutively active mutant of AMPK abolished the high-glucose-induced (30 mmol/l) reduction of GTPCH I, biopeterins, and BH4 but had no effect on GTPCH I mRNA.Furthermore, AICAR or overexpression of AMPK inhibited the high-glucose-enhanced 26S proteasome activity.Consistently, inhibition of the proteasome by MG132 abolished high-glucose-induced reduction of GTPCH I in human umbilical vein endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Diabetes, Department of Medicine, Herald Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

ABSTRACT

Objective: The activation of AMP-activated protein kinase (AMPK) has been reported to improve endothelial function. However, the targets of AMPK in endothelial cells remain poorly defined. The aim of this study was to test whether AMPK suppresses the degradation of GTP-cyclohydrolase (GTPCH I), a key event in vascular endothelial dysfunction in diabetes.

Research design and methods: Both human umbilical vein endothelial cells and aortas isolated from streptozotocin-injected diabetic mice were assayed for phospho-AMPK (Thr172), GTPCH I, tetrahydrobiopterin (BH4), and endothelial functions.

Results: Oral administration of metformin (300 mg x kg(-1) . day(-1), 4 weeks) in streptozotocin-injected mice significantly blunted the diabetes-induced reduction of AMPK phosphorylation at Thr172. Metformin treatment also normalized acetylcholine-induced endothelial relaxation and increased the levels of GTPCH I and BH4. The administration of AICAR, an AMPK activator, or adenoviral overexpression of a constitutively active mutant of AMPK abolished the high-glucose-induced (30 mmol/l) reduction of GTPCH I, biopeterins, and BH4 but had no effect on GTPCH I mRNA. Furthermore, AICAR or overexpression of AMPK inhibited the high-glucose-enhanced 26S proteasome activity. Consistently, inhibition of the proteasome by MG132 abolished high-glucose-induced reduction of GTPCH I in human umbilical vein endothelial cells. Further, aortas isolated from AMPKalpha2(-/-) mice, which exhibited elevated 26S proteasome activity, had reduced levels of GTPCH I and BH4. Finally, either administration of MG132 or supplementation of l-sepiapterin normalized the impaired endothelium-dependent relaxation in aortas isolated from AMPKalpha2(-/-) mice.

Conclusions: We conclude that AMPK activation normalizes vascular endothelial function by suppressing 26S proteasome-mediated GTPCH I degradation in diabetes.

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Activation of AMPK by metformin attenuates diabetes-induced reduction of phospho-AMPK-Thr172 (A), GTPCH I (B), and biopterin (C) levels and improves endothelial function. Control and diabetic mice were fed with metformin (300 mg · kg−1 · day−1) for 4 weeks. Mouse aortas were isolated and assayed for phospho-AMPK, GTPCH I, total biopterins, and BH4 levels, as described in research design and methods. The results were obtained from five mice. *P < 0.05 versus control, #P < 0.05 versus diabetes alone. D: ACh-induced endothelium-dependent relaxation was assayed as described in research design and methods (n = 5 per group, ♣ P < 0.05). l-sepiapterin (10 μmol/l) was added 1 h before the start of the experiments.
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Figure 1: Activation of AMPK by metformin attenuates diabetes-induced reduction of phospho-AMPK-Thr172 (A), GTPCH I (B), and biopterin (C) levels and improves endothelial function. Control and diabetic mice were fed with metformin (300 mg · kg−1 · day−1) for 4 weeks. Mouse aortas were isolated and assayed for phospho-AMPK, GTPCH I, total biopterins, and BH4 levels, as described in research design and methods. The results were obtained from five mice. *P < 0.05 versus control, #P < 0.05 versus diabetes alone. D: ACh-induced endothelium-dependent relaxation was assayed as described in research design and methods (n = 5 per group, ♣ P < 0.05). l-sepiapterin (10 μmol/l) was added 1 h before the start of the experiments.

Mentions: Data published by our group and others demonstrate that AMPK activation exerts beneficial effects by increasing NO bioactivity via an increase in the phosphorylation of eNOS and/or an increase in the anti-oxidant potential of endothelial cells (29–31). Therefore, it was of interest to investigate the effects of hyperglycemia/diabetes on AMPK activity. As the phosphorylation of Thr172 of AMPKα is required for AMPK activity, and because AMPK-Thr172 is closely related to AMPK activity, we first measured the levels of phosphorylated AMPK-Thr172 in mouse aortas isolated from mice with STZ-induced diabetes or nondiabetic mice. As depicted in Fig. 1A, the levels of phospho-AMPK (Thr172) were significantly reduced in diabetic mouse aortas relative to nondiabetic mice. In contrast, the level of AMPKα was not different in diabetic and nondiabetic mice (Fig. 1A). Consistent with our earlier report (9), the levels of GTPCH I were significantly reduced in diabetes relative to the control group (Fig. 1B). In contrast, the levels of eNOS were not altered in diabetic mice or metformin-treated mice (Fig. 1B).


In vivo activation of AMP-activated protein kinase attenuates diabetes-enhanced degradation of GTP cyclohydrolase I.

Wang S, Xu J, Song P, Viollet B, Zou MH - Diabetes (2009)

Activation of AMPK by metformin attenuates diabetes-induced reduction of phospho-AMPK-Thr172 (A), GTPCH I (B), and biopterin (C) levels and improves endothelial function. Control and diabetic mice were fed with metformin (300 mg · kg−1 · day−1) for 4 weeks. Mouse aortas were isolated and assayed for phospho-AMPK, GTPCH I, total biopterins, and BH4 levels, as described in research design and methods. The results were obtained from five mice. *P < 0.05 versus control, #P < 0.05 versus diabetes alone. D: ACh-induced endothelium-dependent relaxation was assayed as described in research design and methods (n = 5 per group, ♣ P < 0.05). l-sepiapterin (10 μmol/l) was added 1 h before the start of the experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712774&req=5

Figure 1: Activation of AMPK by metformin attenuates diabetes-induced reduction of phospho-AMPK-Thr172 (A), GTPCH I (B), and biopterin (C) levels and improves endothelial function. Control and diabetic mice were fed with metformin (300 mg · kg−1 · day−1) for 4 weeks. Mouse aortas were isolated and assayed for phospho-AMPK, GTPCH I, total biopterins, and BH4 levels, as described in research design and methods. The results were obtained from five mice. *P < 0.05 versus control, #P < 0.05 versus diabetes alone. D: ACh-induced endothelium-dependent relaxation was assayed as described in research design and methods (n = 5 per group, ♣ P < 0.05). l-sepiapterin (10 μmol/l) was added 1 h before the start of the experiments.
Mentions: Data published by our group and others demonstrate that AMPK activation exerts beneficial effects by increasing NO bioactivity via an increase in the phosphorylation of eNOS and/or an increase in the anti-oxidant potential of endothelial cells (29–31). Therefore, it was of interest to investigate the effects of hyperglycemia/diabetes on AMPK activity. As the phosphorylation of Thr172 of AMPKα is required for AMPK activity, and because AMPK-Thr172 is closely related to AMPK activity, we first measured the levels of phosphorylated AMPK-Thr172 in mouse aortas isolated from mice with STZ-induced diabetes or nondiabetic mice. As depicted in Fig. 1A, the levels of phospho-AMPK (Thr172) were significantly reduced in diabetic mouse aortas relative to nondiabetic mice. In contrast, the level of AMPKα was not different in diabetic and nondiabetic mice (Fig. 1A). Consistent with our earlier report (9), the levels of GTPCH I were significantly reduced in diabetes relative to the control group (Fig. 1B). In contrast, the levels of eNOS were not altered in diabetic mice or metformin-treated mice (Fig. 1B).

Bottom Line: The administration of AICAR, an AMPK activator, or adenoviral overexpression of a constitutively active mutant of AMPK abolished the high-glucose-induced (30 mmol/l) reduction of GTPCH I, biopeterins, and BH4 but had no effect on GTPCH I mRNA.Furthermore, AICAR or overexpression of AMPK inhibited the high-glucose-enhanced 26S proteasome activity.Consistently, inhibition of the proteasome by MG132 abolished high-glucose-induced reduction of GTPCH I in human umbilical vein endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Diabetes, Department of Medicine, Herald Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

ABSTRACT

Objective: The activation of AMP-activated protein kinase (AMPK) has been reported to improve endothelial function. However, the targets of AMPK in endothelial cells remain poorly defined. The aim of this study was to test whether AMPK suppresses the degradation of GTP-cyclohydrolase (GTPCH I), a key event in vascular endothelial dysfunction in diabetes.

Research design and methods: Both human umbilical vein endothelial cells and aortas isolated from streptozotocin-injected diabetic mice were assayed for phospho-AMPK (Thr172), GTPCH I, tetrahydrobiopterin (BH4), and endothelial functions.

Results: Oral administration of metformin (300 mg x kg(-1) . day(-1), 4 weeks) in streptozotocin-injected mice significantly blunted the diabetes-induced reduction of AMPK phosphorylation at Thr172. Metformin treatment also normalized acetylcholine-induced endothelial relaxation and increased the levels of GTPCH I and BH4. The administration of AICAR, an AMPK activator, or adenoviral overexpression of a constitutively active mutant of AMPK abolished the high-glucose-induced (30 mmol/l) reduction of GTPCH I, biopeterins, and BH4 but had no effect on GTPCH I mRNA. Furthermore, AICAR or overexpression of AMPK inhibited the high-glucose-enhanced 26S proteasome activity. Consistently, inhibition of the proteasome by MG132 abolished high-glucose-induced reduction of GTPCH I in human umbilical vein endothelial cells. Further, aortas isolated from AMPKalpha2(-/-) mice, which exhibited elevated 26S proteasome activity, had reduced levels of GTPCH I and BH4. Finally, either administration of MG132 or supplementation of l-sepiapterin normalized the impaired endothelium-dependent relaxation in aortas isolated from AMPKalpha2(-/-) mice.

Conclusions: We conclude that AMPK activation normalizes vascular endothelial function by suppressing 26S proteasome-mediated GTPCH I degradation in diabetes.

Show MeSH
Related in: MedlinePlus