Glycated proteins stimulate reactive oxygen species production in cardiac myocytes: involvement of Nox2 (gp91phox)-containing NADPH oxidase.
BACKGROUND
Nonenzymatic glycation that results in the production of early-glycation Amadori-modified proteins and advanced-glycation end products may be important in the pathogenesis of diabetic complications. However, the effects of early-glycated proteins, such as glycated serum albumin (Gly-BSA), are poorly defined. In this study, we investigated the effects of Gly-BSA on reactive oxygen species (ROS) production by cardiomyocytes.


RESULTS
Cultured neonatal rat cardiomyocytes were incubated with Gly-BSA or vehicle (bovine serum albumin [BSA]) for up to 48 hours. Gly-BSA dose-dependently increased in situ ROS production (whole-cell dichlorodihydrofluorescein fluorescence), with an optimum effect at 400 microg/mL after 24-hour incubation (152+/-10% versus BSA 100%; P<0.01). Treatment with the NADPH oxidase inhibitor apocynin, a Nox2 (gp91phox) antisense oligonucleotide (Nox2 AS), or the peptide gp91ds-tat significantly reduced Gly-BSA-induced ROS production at 24 hours (68.5+/-2.2%, 61.4+/-8.3%, and 53.2+/-5.4% reduction, respectively). NADPH-dependent activity in cell homogenates was also significantly increased by Gly-BSA at 24 hours (161+/-8% versus BSA) and was inhibited by diphenyleneiodonium, apocynin, NOX2AS, and the protein kinase C inhibitor bisindolylmaleimide I but not by a nitric oxide synthase inhibitor or mitochondrial inhibitors. Furthermore, bisindolylmaleimide I prevented Gly-BSA-stimulated Rac1 translocation, an essential step for NADPH oxidase activation. Gly-BSA-induced increases in ROS were associated with apocynin-inhibitable nuclear translocation of nuclear factor-kappaB and an increase in atrial natriuretic factor mRNA expression.


CONCLUSIONS
Gly-BSA stimulates cardiomyocyte ROS production through a protein kinase C-dependent activation of a Nox2-containing NADPH oxidase, which results in nuclear factor-kappaB activation and upregulation of atrial natriuretic factor mRNA. These findings suggest that early-glycated Amadori products may play a role in the development of diabetic heart disease.
Zhang
Min
M
Cardiovascular Division, King's College London, London, United Kingdom.
Kho
Ay Lin
AL
Anilkumar
Narayana
N
Chibber
Rakesh
R
Pagano
Patrick J
PJ
Shah
Ajay M
AM
Cave
Alison C
AC
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics; D002478:Cells, Cultured; D048909:Diabetes Complications; D006023:Glycoproteins / Q000494:pharmacology*; D006331:Heart Diseases / Q000209:etiology; D006801:Humans; D008562:Membrane Glycoproteins; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D015854:Up-Regulation
D006023:Glycoproteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine; D009320:Atrial Natriuretic Factor; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases