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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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Inhibition of the proteasome and supplementation with l-sepipeterin improves ACh-induced endothelium-dependent relaxation ex vivo. Male AMPKα2−/− mice and their genetic controls (C57BL6 WT mice) were injected with MG132 (5 mg · kg−1 · day−1 for 2 consecutive days) (A) or l-sepiapterin (10 mg · kg−1 · day−1 for 7 days) (B). After the treatments, isolated mouse aortas assayed for ACh-induced endothelium-dependent relaxation (n = 5 per group, ♣ P < 0.05). C: Proposed scheme.
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Figure 7: Inhibition of the proteasome and supplementation with l-sepipeterin improves ACh-induced endothelium-dependent relaxation ex vivo. Male AMPKα2−/− mice and their genetic controls (C57BL6 WT mice) were injected with MG132 (5 mg · kg−1 · day−1 for 2 consecutive days) (A) or l-sepiapterin (10 mg · kg−1 · day−1 for 7 days) (B). After the treatments, isolated mouse aortas assayed for ACh-induced endothelium-dependent relaxation (n = 5 per group, ♣ P < 0.05). C: Proposed scheme.

Mentions: To establish a cause-effect relationship between proteasome-dependent GTPCH I degradation and impaired endothelium-dependent relaxation in AMPKα2−/− mice, we injected AMPKα2−/− mice with MG132 (5 mg · kg−1 · day−1 for 2 days, intraperitoneally) or l-sepiapterin (10 mg · kg−1 · day−1 for 7 days, intraperitoneally). After the treatments, mouse aortas were isolated and assayed for endothelium-dependent and endothelium-independent relaxation. As shown in Fig. 7A, the maximal relaxation (Emax) induced by ACh is 83.63 ± 7.60% in WT aortas. However, Emax of ACh is only 60.9 ± 5.5 in AMPKα2−/− aortas (P < 0.05 vs. WT). MG132 did not alter the ACh-induced endothelium-dependent relaxation in WT mice but improved the relaxation in AMPKα2−/− mice. As shown in Fig. 7B, l-sepiapterin also mitigated the decrease of ACh-induced Emax in AMPKα2−/− mice (73.82 ± 9.50% vs. AMPKα2−/−, P < 0.05). SNP-induced, endothelium-independent relaxations in aortas were comparable among three groups (data not shown). Taken together, these results support the idea that the impaired endothelial function of AMPKα2−/− mice is because of proteasome-dependent GTPCH I degradation.


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)

Inhibition of the proteasome and supplementation with l-sepipeterin improves ACh-induced endothelium-dependent relaxation ex vivo. Male AMPKα2−/− mice and their genetic controls (C57BL6 WT mice) were injected with MG132 (5 mg · kg−1 · day−1 for 2 consecutive days) (A) or l-sepiapterin (10 mg · kg−1 · day−1 for 7 days) (B). After the treatments, isolated mouse aortas assayed for ACh-induced endothelium-dependent relaxation (n = 5 per group, ♣ P < 0.05). C: Proposed scheme.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Inhibition of the proteasome and supplementation with l-sepipeterin improves ACh-induced endothelium-dependent relaxation ex vivo. Male AMPKα2−/− mice and their genetic controls (C57BL6 WT mice) were injected with MG132 (5 mg · kg−1 · day−1 for 2 consecutive days) (A) or l-sepiapterin (10 mg · kg−1 · day−1 for 7 days) (B). After the treatments, isolated mouse aortas assayed for ACh-induced endothelium-dependent relaxation (n = 5 per group, ♣ P < 0.05). C: Proposed scheme.
Mentions: To establish a cause-effect relationship between proteasome-dependent GTPCH I degradation and impaired endothelium-dependent relaxation in AMPKα2−/− mice, we injected AMPKα2−/− mice with MG132 (5 mg · kg−1 · day−1 for 2 days, intraperitoneally) or l-sepiapterin (10 mg · kg−1 · day−1 for 7 days, intraperitoneally). After the treatments, mouse aortas were isolated and assayed for endothelium-dependent and endothelium-independent relaxation. As shown in Fig. 7A, the maximal relaxation (Emax) induced by ACh is 83.63 ± 7.60% in WT aortas. However, Emax of ACh is only 60.9 ± 5.5 in AMPKα2−/− aortas (P < 0.05 vs. WT). MG132 did not alter the ACh-induced endothelium-dependent relaxation in WT mice but improved the relaxation in AMPKα2−/− mice. As shown in Fig. 7B, l-sepiapterin also mitigated the decrease of ACh-induced Emax in AMPKα2−/− mice (73.82 ± 9.50% vs. AMPKα2−/−, P < 0.05). SNP-induced, endothelium-independent relaxations in aortas were comparable among three groups (data not shown). Taken together, these results support the idea that the impaired endothelial function of AMPKα2−/− mice is because of proteasome-dependent GTPCH I degradation.

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