Limits...
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.

Show MeSH

Related in: MedlinePlus

AMPK activation does not alter GTPCH I expression in HUVECs. A: HUVECs were treated with high glucose (HG) for 24 h in the presence or absence of AICAR (2 mmol/l). Cells were subjected to perform RT-PCR analysis for GTPCH I mRNA expression. The picture shown is a representative of three blots of three independent experiments. B: HUVECs were treated with metformin, AICAR, or high glucose in the presence or absence of cycloheximide (100 μg/ml) or actinomycin D (10 μg/ml) for 24 h, and GTPCH I protein expression was then detected by Western blot. The blot shown is a representative of three independent experiments. *P < 0.05 versus control, #P < 0.05 versus cycloheximide alone, ¥P < 0.05 versus actinomycin D alone, NS: P > 0.05. NG, normal glucose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2712774&req=5

Figure 4: AMPK activation does not alter GTPCH I expression in HUVECs. A: HUVECs were treated with high glucose (HG) for 24 h in the presence or absence of AICAR (2 mmol/l). Cells were subjected to perform RT-PCR analysis for GTPCH I mRNA expression. The picture shown is a representative of three blots of three independent experiments. B: HUVECs were treated with metformin, AICAR, or high glucose in the presence or absence of cycloheximide (100 μg/ml) or actinomycin D (10 μg/ml) for 24 h, and GTPCH I protein expression was then detected by Western blot. The blot shown is a representative of three independent experiments. *P < 0.05 versus control, #P < 0.05 versus cycloheximide alone, ¥P < 0.05 versus actinomycin D alone, NS: P > 0.05. NG, normal glucose.

Mentions: Increased levels of GTPCH I could be because of the transcriptional activation of the GTPCH I gene or attributable to the decrease of GTPCH I protein degradation. We first determined whether AMPK activation altered GTPCH I mRNA levels in HUVECs. As shown in Fig. 4A, treatment of HUVECs with AICAR (2 mmol/l) for 24 h did not alter the levels of GTPCH I mRNA in HUVECs exposed to normal glucose or high glucose. In addition, neither cycloheximide nor actinomycin D altered the levels of GTPCH I protein caused by AICAR or metformin (Fig. 4B). Neither cycloheximide nor actinomycin D altered the high-glucose–induced reduction of GTPCH I levels in HUVECs (Fig. 4B). Taken together, our data suggest that the increase of GTPCH I protein levels caused by AMPK activation is independent of transcriptional activation of the GTPCH I gene.


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)

AMPK activation does not alter GTPCH I expression in HUVECs. A: HUVECs were treated with high glucose (HG) for 24 h in the presence or absence of AICAR (2 mmol/l). Cells were subjected to perform RT-PCR analysis for GTPCH I mRNA expression. The picture shown is a representative of three blots of three independent experiments. B: HUVECs were treated with metformin, AICAR, or high glucose in the presence or absence of cycloheximide (100 μg/ml) or actinomycin D (10 μg/ml) for 24 h, and GTPCH I protein expression was then detected by Western blot. The blot shown is a representative of three independent experiments. *P < 0.05 versus control, #P < 0.05 versus cycloheximide alone, ¥P < 0.05 versus actinomycin D alone, NS: P > 0.05. NG, normal glucose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: AMPK activation does not alter GTPCH I expression in HUVECs. A: HUVECs were treated with high glucose (HG) for 24 h in the presence or absence of AICAR (2 mmol/l). Cells were subjected to perform RT-PCR analysis for GTPCH I mRNA expression. The picture shown is a representative of three blots of three independent experiments. B: HUVECs were treated with metformin, AICAR, or high glucose in the presence or absence of cycloheximide (100 μg/ml) or actinomycin D (10 μg/ml) for 24 h, and GTPCH I protein expression was then detected by Western blot. The blot shown is a representative of three independent experiments. *P < 0.05 versus control, #P < 0.05 versus cycloheximide alone, ¥P < 0.05 versus actinomycin D alone, NS: P > 0.05. NG, normal glucose.
Mentions: Increased levels of GTPCH I could be because of the transcriptional activation of the GTPCH I gene or attributable to the decrease of GTPCH I protein degradation. We first determined whether AMPK activation altered GTPCH I mRNA levels in HUVECs. As shown in Fig. 4A, treatment of HUVECs with AICAR (2 mmol/l) for 24 h did not alter the levels of GTPCH I mRNA in HUVECs exposed to normal glucose or high glucose. In addition, neither cycloheximide nor actinomycin D altered the levels of GTPCH I protein caused by AICAR or metformin (Fig. 4B). Neither cycloheximide nor actinomycin D altered the high-glucose–induced reduction of GTPCH I levels in HUVECs (Fig. 4B). Taken together, our data suggest that the increase of GTPCH I protein levels caused by AMPK activation is independent of transcriptional activation of the GTPCH I gene.

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