Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy
Introduction
Several experimental, epidemiological, and clinical studies support the view that oxidative stress plays a significant role in the development of vascular complications in diabetics [1], [2]. The importance of reactive oxygen species (ROS) in the pathogenesis of diabetes and its complications are well recognized and a considerable body of evidence implicates ROS in defective cellular signaling [3], [4]. However, the molecular mechanisms by which ROS alter cellular signaling are not well understood. Reactive thiols on cysteine (Cys) residues of selected proteins are among the most sensitive sites to be modified by ROS. Oxidants react with these redox-sensitive thiols to form thiyl radicals, which subsequently react with other thiols to form mixed-disulfide bonds. In mammalian cells, glutathione is the most abundant low molecular thiol, and hence the most likely to bind to protein thiols to form mixed-disulfides, a process termed S-glutathionylation [5], [6].
Since a number of functionally critical proteins within the cell possess accessible Cys residues, glutathionylation may be considered an important post-translational modification in the pathogenesis of complex diseases such as diabetes. Therefore, we hypothesize that, under conditions of increased oxidative stress and changes in glutathione levels, many critical proteins are liable to undergo glutathionylation in patients with diabetes and its associated complications. In recent years, glutathionylated proteins including HbSSG have been investigated as possible biomarkers of oxidative stress using the HPLC or LC-MS detection systems [6], [7], [8], [9], [10]. Since the blood concentrations of glutathionylated proteins may reflect alterations in redox signaling and oxidation status [10], [11], in the present study, we have used erythrocytes as a cellular model to test HbSSG as a possible biomarker of increased oxidative stress.
Section snippets
Methods
The study group was comprised of a total of 130 subjects, which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. The diabetic subjects were selected from out-patients attending the Dr. Mohans' M.V Diabetes Specialties Centre (MVDSC), a tertiary referral center for diabetes care at Chennai (formerly Madras) in Southern India. Control subjects were recruited from the on-going Chennai Urban Rural
Results
Table 1 shows the clinical characteristics of the study groups. Diabetic subjects without microangiopathy had higher systolic blood pressure compared to control subjects. Diabetic subjects with microangiopathy had significantly higher systolic and diastolic blood pressures, serum triglyceride, and LDL-C levels compared to control subjects.
Fig. 1a shows an ESI mass spectrum of a non-diabetic (control) subject's hemoglobin. Deconvolution of the charge states (transformed mass scale) distinctively
Discussion
Currently, there is a growing awareness of the importance of oxidants as signaling molecules that directly impact the function of tissues in a regulatory fashion by altering the structure of proteinaceous cysteinyl thiols. Multiple modes of protein–cysteine oxidation, such as S-thiolation, S-nitrosylation, sulfenic acid, sulfinic acid, sulfenyl-amide formation, reactive oxidized lipid derivates, and intra- and intermolecular protein disulfides are already known to be important in redox
Acknowledgments
This work was supported by a research grant from the Department of Science and Technology (DST), New Delhi, India. The mass spectrometry facilities at the Indian Institute of Science are funded by a program support grant from the Department of Biotechnology, India. S.S. acknowledges a postdoctoral fellowship from the Department of Biotechnology, India. We thank the Chennai Willington Corporation Foundation for their support for the CURES field studies. This is paper No. 15 from the CURES study.
References (52)
A simple fluorometric assay for lipoperoxide in blood plasma
Biochem. Med.
(1976)- et al.
Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis
J. Biol. Chem.
(2004) - et al.
Antioxidant status, lipid peroxidation and nitric oxide end products in patients of type 2 diabetes mellitus with nephropathy
Clin. Biochem.
(2003) - et al.
Glutathione depletion in the lens of galactsaemic and diabetic rats
Exp. Eye Res.
(1988) - et al.
PEDF derived from glial Muller cells: a possible regulator of retinal angiogenesis
Exp. Cell Res.
(2004) - et al.
Glucose-induced oxidative stress in mesangial cells
Kidney Int.
(2002) - et al.
Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation
Biochem. Biophys. Res. Commun.
(2002) - et al.
S-glutathionylation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells
J. Biol. Chem.
(2004) - et al.
S-glutathionylation of NF-kappa B subunit p50
Methods Enzymol.
(2002) - et al.
S-glutathionylation decreases Mg2+ inhibition and S-nitrosylation enhances Ca2+ activation of RyR1 channels
J. Biol. Chem.
(2003)
Regulation of protein tyrosine phosphatase 1B in intact cells by S-nitrosothiols
Arch. Biochem. Biophys.
Regulation of cAMP-dependent protein kinase activity by glutathionylation
J. Biol. Chem.
Modulation of aldose reductase activity through S-thiolation by physiological thiols
Chem. Biol. Interact.
Reversible glutathionylation of complex I increases mitochondrial superoxide formation
J. Biol. Chem.
Oxidative stress and diabetic vascular complications
Curr. Diabetes Rep.
Antioxidant therapy in diabetic complications: what is new?
Curr. Vasc. Pharmacol.
Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited
Arterioscler. Thromb. Vasc. Biol.
Glucose toxicity in beta cells: type 2 diabetes, good radicals gone bad, and the glutathione connection
Diabetes
Oxidants, oxidative stress and the biology of ageing
Nature
Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry
Clin. Chem.
The potential use of glutathionyl hemoglobin as a clinical marker of oxidative stress
Clin. Chem.
Characterization of a novel hemoglobin-glutathione adduct that is elevated in diabetic patients
Mol. Med.
Reversible cysteine-targeted oxidation of proteins during renal oxidative stress
J. Am. Soc. Nephrol.
Protein glutathionylation in erythrocytes
Clin. Chem.
Oxidation state of glutathione in the erythrocyte
Clin. Sci. (London)
The Chennai Urban Rural Epidemiology Study (CURES)—Study design and methodology (urban component) (CURES-I)
J. Assoc. Phys. India
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