Effects of Irbesartan on Intracellular Antioxidant Enzyme Expression and Activity in Adolescents and Young Adults With Early Diabetic Angiopathy FRANCESCO CHIARELLI, MD, PHD 1 DANIELE DI MARZIO, MD 1 FRANCESCA SANTILLI, MD 1 ANGELIKA MOHN, MD 1 ANNALISA BLASETTI, MD 1 FRANCESCO CIPOLLONE, MD 2 ANDREA MEZZETTI, MD, PHD 2 ALBERTO VERROTTI, MD, PHD 1 OBJECTIVE — Defective intracellular antioxidant enzyme production (IAP) has been dem- onstrated in adults with diabetic nephropathy. The objective of this study was to evaluate the effects of irbesartan, an angiotensin II receptor antagonist, on IAP in adolescents and young adults with type 1 diabetes and early signs of retinopathy and nephropathy. RESEARCH DESIGN AND METHODS — This prospective, matched case-control study was conducted between November 2001 and December 2002 among 14 type 1 diabetic patients with early signs of angiopathy (ages 14 –21 years), 11 type 1 diabetic patients without angiopathy (ages 12–22 years), and 10 healthy volunteers (ages 16 –22 years). Skin fibroblasts were obtained by skin biopsies from the anterior part of the forearm and cultured in Dulbecco’s modified Eagle’s medium. The activity and mRNA expression of CuZn superoxide dismutase (CuZnSOD), Mn superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase (GPX) were measured before and after 6 months of treatment with irbesartan (150 mg/day); on both occasions, antioxidant enzyme activity was evaluated at different glucose concentrations (5 and 22 mmol/l). RESULTS — At a normal glucose concentration (5 mmol/l), the activity and mRNA expres- sion of CuZnSOD (0.50  0.21 units/mg protein, 4.4  1.5 mRNA/glyceraldehyde-3-phosphate dehydrogenase), MnSOD (0.26  0.04 units/mg protein, 0.08  0.07 mRNA), CAT (0.32  0.08 units/mg protein, 4.8  1.3 mRNA), and GPX (0.53  0.09 units/mg protein, 2.2  0.9 mRNA) were not different among the three groups (only values of diabetic subjects with angi- opathy are shown). At high glucose concentrations, the activity and mRNA expression of CuZnSOD increased similarly in all groups (diabetic subjects with angiopathy: 0.93  0.26 units/mg protein, 9.4  2.1 mRNA); that of CAT and GPX increased in only control subjects and diabetic subjects without angiopathy (diabetic subjects with angiopathy: 0.33  0.09 units/mg protein and 5.0  1.4 mRNA; 0.54  0.10 units/mg protein and 2.3  1.0 mRNA, respectively). MnSOD did not change in any group. Treatment with irbesartan in adolescents with diabetic angiopathy was able to restore CAT and GPX activity and mRNA expression after exposure to high glucose concentrations. Markers of oxidative stress (serum malondialdehyde, fluorescent products of lipid peroxidation, monocyte chemoattractant protein-1, and 8-isoprostanes pros- taglandin F 2 ) were significantly reduced after treatment with irbesartan. CONCLUSIONS — Adolescents and young adults with early signs of diabetic angi- opathy have defective intracellular antioxidant enzyme production and activity. Treatment with irbesartan can substantially improve the activity and production of these enzymes in skin fibroblasts. Diabetes Care 28:1690 –1697, 2005 I t has recently been shown that oxida- tive stress may play a role in the patho- genesis of diabetic vascular complications (1). Hyperglycemia can in- duce increased production of reactive ox- ygen metabolites and species. Elevated glucose concentrations may also increase the levels of oxygen radical scavenging enzymes in cultured endothelial cells (1) and the kidney of rats with streptozoto- cin-induced diabetes (1). The formation of oxygen-derived free radicals and the activation of oxidative stress through nonenzymatic glycation of proteins, auto- oxidative glycation, activation of protein kinase C, and the increased polyol path- way may be induced by hyperglycemia. In normal subjects, exposure to high glucose concentrations induces an anti- oxidant defensive mechanism in skin fi- broblasts; in adult type 1 diabetic subjects with macroalbuminuria and overt ne- phropathy, this defensive mechanism is markedly impaired (2). A defective anti- oxidant response to hyperglycemia has been recently demonstrated in peripheral blood mononuclear cells of patients with type 1 diabetes and diabetic nephropathy (3). Recently, we demonstrated that vita- min E supplementation (600 mg, b.i.d. for 3 months) is unable to significantly modify antioxidant mechanisms in skin fibroblasts of adolescents and young adults with type 1 diabetes and angiopa- thy (retinopathy or nephropathy) (4). Irbesartan is a potent and selective an- giotensin II (ANG-II) subtype 1 receptor antagonist widely used in adult patients ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● From the 1 Department of Pediatrics, University of Chieti, Chieti, Italy; and the 2 Department of Medicine, University of Chieti, Chieti, Italy. Address correspondence and reprint requests to Francesco Chiarelli, Department of Pediatrics, University of Chieti, Ospedale Policlinico, Via dei Vestini, 5, I-66100 Chieti, Italy. E-mail: chiarelli@unich.it. Received for publication 26 January 2005 and accepted in revised form 28 March 2005. Abbreviations: AER, albumin excretion rate; ANG-II, angiotensin II; CAT, catalase; CuZnSOD, CuZn superoxide dismutase; DMEM, Dulbecco’s modified Eagle’s medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPX, glutathione peroxidase; MCP-1, monocyte chemoattractant protein 1; MnSOD, Mn superoxide dismutase; PGF 2a , prostaglandin F 2a ; RAS, renin-angiotensin system; SOD, superoxide dis- mutase; SSC, standard sodium citrate. A table elsewhere in this issue shows conventional and Syste `me International (SI) units and conversion factors for many substances. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Pathophysiology/Complications O R I G I N A L A R T I C L E 1690 DIABETES CARE, VOLUME 28, NUMBER 7, JULY 2005