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Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus.

ABSTRACT: HMG-CoA reductase inhibitors have been shown to upregulate GTP cyclohydrolase I (GTPCH-I), the key enzyme for tetrahydrobiopterin de novo synthesis and to normalize tetrahydrobiopterin levels in hyperglycemic endothelial cells. We sought to determine whether in vivo treatment with the HMG-CoA reductase inhibitor atorvastatin is able to upregulate the GTPCH-I, to recouple eNOS and to normalize endothelial dysfunction in an experimental model of diabetes mellitus.In male Wistar rats, diabetes was induced by streptozotocin (STZ, 60 mg/kg). In STZ rats, atorvastatin feeding (20 mg/kg/d, 7 weeks), normalized vascular dysfunction as analyzed by isometric tension studies, levels of circulating endothelial progenitor cells (FACS-analysis), superoxide formation (assessed by lucigenin-enhanced chemiluminescence and dihydroethidium staining), vascular levels of the phosphorylated vasodilator-stimulated phosphoprotein (P-VASP), tyrosine nitration of the prostacyclin synthase, expression of GTPCH-I, dihydrofolate reductase and eNOS, translocation of regulatory NADPH oxidase subunits rac1, p47phox and p67phox (assessed by Western blot) and vascular tetrahydrobiopterin levels as measured by HPLC. Dihydroethidine staining revealed that the reduction of vascular superoxide was at least in part due to eNOS recoupling.HMG-CoA reductase inhibition normalizes endothelial function and reduces oxidative stress in diabetes by inhibiting activation of the vascular NADPH oxidase and by preventing eNOS uncoupling due to an upregulation of the key enzyme of tetrahydrobiopterin synthesis, GTPCH-I.

SUBMITTER: Wenzel P

PROVIDER: S-EPMC2889614 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus.

Wenzel Philip P Daiber Andreas A Oelze Matthias M Brandt Moritz M Closs Ellen E Xu Jian J Thum Thomas T Bauersachs Johann J Ertl Georg G Zou Ming-Hui MH Förstermann Ulrich U Münzel Thomas T

Atherosclerosis 20071203 1

<h4>Objective</h4>HMG-CoA reductase inhibitors have been shown to upregulate GTP cyclohydrolase I (GTPCH-I), the key enzyme for tetrahydrobiopterin de novo synthesis and to normalize tetrahydrobiopterin levels in hyperglycemic endothelial cells. We sought to determine whether in vivo treatment with the HMG-CoA reductase inhibitor atorvastatin is able to upregulate the GTPCH-I, to recouple eNOS and to normalize endothelial dysfunction in an experimental model of diabetes mellitus.<h4>Methods and ...[more]

PMID: 18061195

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Project description:HMG-CoA reductase (HMGR) is the target of statins, cholesterol-lowering drugs prescribed to millions of patients worldwide. More recent research indicates that HMGR could be a useful target in the development of antimicrobial agents. Over the last seven decades, researchers have proposed a series of increasingly complex reaction mechanisms for this biomedically important enzyme. The maturation of the mechanistic proposals for HMGR have paralleled advances in a diverse set of research areas, such as molecular biology and computational chemistry. Thus, the development of the HMGR mechanism provides a useful case study for following the advances in state-of-the-art methods in enzyme mechanism research. Similarly, the questions raised by these mechanism proposals reflect the limitations of the methods used to develop them. The mechanism of HMGR, a four-electron oxidoreductase, is unique and far more complex than originally thought. The reaction contains multiple chemical steps, coupled to large-scale domain motions of the homodimeric enzyme. The first proposals for the HMGR mechanism were based on kinetic and labeling experiments, drawing analogies to the mechanism of known dehydrogenases. Advances in molecular biology and bioinformatics enabled researchers to use site-directed mutagenesis experiments and protein sequencing to identify catalytically important glutamate, aspartate, and histidine residues. These studies, in turn, have generated new and more complicated mechanistic proposals. With the development of protein crystallography, researchers solved HMGR crystal structures to reveal an unexpected lysine residue at the center of the active site. The many crystal structures of HMGR led to increasingly complex mechanistic proposals, but the inherent limitations of the protein crystallography left a number of questions unresolved. For example, the protonation state of the glutamate residue within the active site cannot be clearly determined from the crystal structure. The differing protonation state of this residue leads to different proposed mechanisms for the enzyme. As computational analysis of large biomolecules has become more feasible, the application of methods such as hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to the HMGR mechanism have led to the most detailed mechanistic proposal yet. As these methodologies continue to improve, they prove to be very powerful for the study of enzyme mechanisms in conjunction with protein crystallography. Nevertheless, even the most current mechanistic proposal for HMGR remains incomplete due to limitations of the current computational methodologies. Thus, HMGR serves as a model for how the combination of increasingly sophisticated experimental and computational methods can elucidate very complex enzyme mechanisms.

Dataset Information

Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus.

Publications

Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus.

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