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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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Related in: MedlinePlus

Activation of AMPK is required for the AICAR/metformin-restored reduction of GTPCH I caused by high-glucose exposure in HUVECs. HUVECs were treated with high glucose (HG) (30 mmol/l d-glucose) or hyperosmatic medium (5 mmol/l glucose plus 25 mmol/l l-glucose) for 24 h in the presence or absence of AICAR (2 mmol/l). HUVECs were collected and assayed for the levels of P-AMPK and GTPCH I protein by Western blot (A) and total biopterins and BH4 (B) (n = 5 per group, *P < 0.05 vs. control, #P < 0.05 versus high glucose alone). C and D: Overexpression of AMPK-DN abolishes metformin's effects on GTPCH I, biopterins, and BH4. HUVECs were transfected with ad-GFP or ad-AMPK-DN for 24 h and then incubated with or without metformin (2 mmol/l) for 8 h. The results are a summary of three independent experiments. *P < 0.05 versus GFP alone, #P < 0.05 versus GFP plus metformin, NS: P > 0.05. HO, hyperosmolarity control; NG, normal glucose.
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Figure 2: Activation of AMPK is required for the AICAR/metformin-restored reduction of GTPCH I caused by high-glucose exposure in HUVECs. HUVECs were treated with high glucose (HG) (30 mmol/l d-glucose) or hyperosmatic medium (5 mmol/l glucose plus 25 mmol/l l-glucose) for 24 h in the presence or absence of AICAR (2 mmol/l). HUVECs were collected and assayed for the levels of P-AMPK and GTPCH I protein by Western blot (A) and total biopterins and BH4 (B) (n = 5 per group, *P < 0.05 vs. control, #P < 0.05 versus high glucose alone). C and D: Overexpression of AMPK-DN abolishes metformin's effects on GTPCH I, biopterins, and BH4. HUVECs were transfected with ad-GFP or ad-AMPK-DN for 24 h and then incubated with or without metformin (2 mmol/l) for 8 h. The results are a summary of three independent experiments. *P < 0.05 versus GFP alone, #P < 0.05 versus GFP plus metformin, NS: P > 0.05. HO, hyperosmolarity control; NG, normal glucose.

Mentions: As metformin treatment increased the amount of GTPCH I and BH4 in diabetic aortas, we reasoned that metformin improves endothelial function through the inhibition of diabetes-induced GTPCH I degradation. To test this hypothesis, cultured HUVECs were exposed to normal glucose (5 mmol/l), high glucose (30 mmol/l d-glucose), or hyperosmotic control (5 mmol/l d-glucose plus 25 mmol/l l-glucose) in the presence or absence of AICAR (2 mmol/l), an AMPK activator. High glucose did not alter AMPK and eNOS expressions (Fig. 2A). However, consistent with the reduction of both total biopterins and BH4 levels, the levels of AMPK-Thr172 were markedly reduced in high-glucose–exposed HUVECs (Fig. 2A and B). Consistent with our previous report, neither total biopterins nor BH4 levels were altered in the hyperosmatic control group, compared to HUVECs in normal glucose (Fig. 2B). As expected, AICAR treatment increased the level of AMPK phosphorylation at Thr172 in HUVECs exposed to either normal glucose or high glucose without altering AMPK and eNOS expressions (Fig. 2A).


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 is required for the AICAR/metformin-restored reduction of GTPCH I caused by high-glucose exposure in HUVECs. HUVECs were treated with high glucose (HG) (30 mmol/l d-glucose) or hyperosmatic medium (5 mmol/l glucose plus 25 mmol/l l-glucose) for 24 h in the presence or absence of AICAR (2 mmol/l). HUVECs were collected and assayed for the levels of P-AMPK and GTPCH I protein by Western blot (A) and total biopterins and BH4 (B) (n = 5 per group, *P < 0.05 vs. control, #P < 0.05 versus high glucose alone). C and D: Overexpression of AMPK-DN abolishes metformin's effects on GTPCH I, biopterins, and BH4. HUVECs were transfected with ad-GFP or ad-AMPK-DN for 24 h and then incubated with or without metformin (2 mmol/l) for 8 h. The results are a summary of three independent experiments. *P < 0.05 versus GFP alone, #P < 0.05 versus GFP plus metformin, NS: P > 0.05. HO, hyperosmolarity control; NG, normal glucose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Activation of AMPK is required for the AICAR/metformin-restored reduction of GTPCH I caused by high-glucose exposure in HUVECs. HUVECs were treated with high glucose (HG) (30 mmol/l d-glucose) or hyperosmatic medium (5 mmol/l glucose plus 25 mmol/l l-glucose) for 24 h in the presence or absence of AICAR (2 mmol/l). HUVECs were collected and assayed for the levels of P-AMPK and GTPCH I protein by Western blot (A) and total biopterins and BH4 (B) (n = 5 per group, *P < 0.05 vs. control, #P < 0.05 versus high glucose alone). C and D: Overexpression of AMPK-DN abolishes metformin's effects on GTPCH I, biopterins, and BH4. HUVECs were transfected with ad-GFP or ad-AMPK-DN for 24 h and then incubated with or without metformin (2 mmol/l) for 8 h. The results are a summary of three independent experiments. *P < 0.05 versus GFP alone, #P < 0.05 versus GFP plus metformin, NS: P > 0.05. HO, hyperosmolarity control; NG, normal glucose.
Mentions: As metformin treatment increased the amount of GTPCH I and BH4 in diabetic aortas, we reasoned that metformin improves endothelial function through the inhibition of diabetes-induced GTPCH I degradation. To test this hypothesis, cultured HUVECs were exposed to normal glucose (5 mmol/l), high glucose (30 mmol/l d-glucose), or hyperosmotic control (5 mmol/l d-glucose plus 25 mmol/l l-glucose) in the presence or absence of AICAR (2 mmol/l), an AMPK activator. High glucose did not alter AMPK and eNOS expressions (Fig. 2A). However, consistent with the reduction of both total biopterins and BH4 levels, the levels of AMPK-Thr172 were markedly reduced in high-glucose–exposed HUVECs (Fig. 2A and B). Consistent with our previous report, neither total biopterins nor BH4 levels were altered in the hyperosmatic control group, compared to HUVECs in normal glucose (Fig. 2B). As expected, AICAR treatment increased the level of AMPK phosphorylation at Thr172 in HUVECs exposed to either normal glucose or high glucose without altering AMPK and eNOS expressions (Fig. 2A).

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