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.
