BENFOTIAMINE PREVENTS THE CONSEQUENCES OF HYPERGLYCEMIA-INDUCED MITOCHONDRIAL OVERPRODUCTION OF REACTIVE OXYGEN SPECIES, AND EXPERIMENTAL DIABETIC RETINOPATHY

R. Bergfeld, T. Matsumara, X. Du, M. Brownlee

University Hospital Mannheim,
University of Heidelberg, German,
Justus-Liebig-University Giessen, Germany
Albert-Einstein College of Medicine, New York, USA

Abstract

Background and Aims: Vascular complications are the main cause of morbidity and mortality in diabetes mellitus. Four seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C (PKC) isoforms, increased formation of glucose-derived advanced glycation end products; increased glucose flux through the aldose reductase pathway, and increased flux through the hexosamine pathway. Hyperglycemia increases reactive oxygen species (ROS) production inside cultured bovine aortic endothelial cells. ROS activate aldose reductase, activate PKC, induce advanced glycation end product formation, activate the hexosamine pathway, and activate the pleiotropic transcription factor nuclear factor-kappa B (NFkB). The thiamine pro-drug benfotiamine inhibits the formation of AGEs in target tissues of diabetic microangiopathy.

Materials and Methods: Using bovine aortic endothelial cells, we studied the effect of benfotiamin on intracellular AGE-formation, flux through the hexosamine pathway, activation of protein kinase C, and activation of NFkB. Benfotiamine was added to cells in high glucose media at a final concentration of 50 µM. Media was changed daily for 7 days. AGE-formation was determined Cell by dot blot technique, and complexes were visualized using an ECF kit (Amersham). Cell extracts were analysed on an HPLC system as described previously (PNAS 2000 97: 12222-12226). For NFkB determination cells were incubated in low glucose, high glucose, high glucose + benfotiamine for 6 hrs. NFkB was determined by a fluorescence in situ DNA-protein binding assay and fluorescence/cell was determined using Scanalytics. PKC assay was performed after cells were incubated for 7 days as described above. The cells were analysed using a PKC assay system from Life Technologies. Additionally, diabetic rats (i.v. injection of streptozotocin 65 mg/kg body weight) were treated with benfotiamin (80 mg/kg weight) for 36 weeks, and diabetic retinopathy was assessed using quantitative retinal analysis of digest preparations for the development of acellular capillaries. Age-matched non-diabetic and untreated diabetic rats served as controls.

Results: Benfotiamine decreases AGE formation by 60% using quantitative immunoblotting. UDP-GlcNAc was decreased 50% by benfotiamine using the previous conditions. NFKB was decreased 85% to below control levels. Benfotiamine decreased membrane PKC 55% to control levels. Chronic treatment of benfotiamine was well tolerated and did not result in significant changes of metabolic parameters. The development of acellular capillaries was reduced by benfotiamine-treatment from 72.5 ± 11.16 acellular capillary segments/mm2 of retinal area in diabetic rats to 29.64 ± 4.48 acellular capillary segments/mm2 in benfotiamine-treated rats (p < 0.001).

Conclusion: These data suggest that treatment with benfotiamine may be an effective approach to prevent the development of diabetic complications.