INTRACELLULAR GLYCATION AND DIABETIC COMPLICATIONS

DESCRIPTION This revised
application provides a summary of work accomplished during the previous
funding period involving the role of glucose derived nonenzymatic
modifications of extracellular matrix in the pathogenesis of diabetic
microvascular disease. It also provides preliminary data about the
potential role of intracellular glycation in the pathogenesis of
diabetic microvascular disease, and outlines in detail experiments to
evaluate this unexplored mechanism. The hypothesis to be examined is
that nonenzymatic glycation occurs intracellularly, where glucose
derived sugars react with proteins up to 200 times faster than glucose.
It is postulated that excessive intracellular glycation in vascular
cells would modify the function of proteins involved in the regulation
of normal cell growth and basement membrane production. bFGF has been
selected as a model protein for intensive investigation (a) because it
is stored intracellularly rather than secreted, and (b) because it plays
an important role in microvascular homeostasis. To determine the effect of elevated extracellular glucose concentration
on intracellular glycated protein and bFGF concentration, proteins from
the endothelial cell line GM7373 will be analyzed using boronate
affinity chromatography, Western blotting, and if required, immuno-PCR.
To determine the effect of increased glucose 6-phosphate and other
reactive glycolytic intermediates on intracellular glycation, expression
vectors will be used to derive GM7373 endothelial cell clones stably
transfected with the high Km beta-cell glucokinase gene. Clones will
be characterized by immunoblotting, glucose 6-phosphate and glucokinase
assays, and glycated proteins will be evaluated as described above. To
evaluate the biologic consequences of intracellular glycation of bFGF,
binding affinities will be evaluated using standard methods for
radiolabelling, glycation, protein purification, and competitive
radioreceptor assay. Characterization of glycated bFGF's biological
activities will be assessed in standard mitogenesis and plasminogen
activator generating assays. To characterize the specific chemical
nature of intracellular glycation, amounts of intracellularly glycated
bFGF required for analysis will be produced using CHO cells transfected
with the amplifiable vector pEE14 containing bFGF cDNA. Peptide
mapping, sequencing, and fast-atom bombardment mass spectroscopy will
be used to analyze peptide and carbohydrate of intracellularly glycated
bFGF.