Effects of nonenzymatic glycation and fatty acids on functional properties of human albumin.

Human serum albumin nonenzymatically condenses with glucose to form stable Amadori adducts that are increased with the hyperglycemia of diabetes. The present study evaluated the influence of fatty acids, which are major endogenous ligands, on albumin glycation and of glycation on albumin conformation and exogenous ligand binding. Physiologic concentrations of palmitate, oleate, and linoleate reduced the ability of albumin to form glucose adducts, whereas glycation decreased intrinsic fluorescence, lowered the affinity for dansylsarcosine, and diminished the fatty acid-induced increase in limiting fluorescence of protein-bound warfarin that was observed with nonglycated albumin.

Amelioration of diabetes-associated abnormalities in the vitreous fluid by an inhibitor of albumin glycation.

Purpose: Albumin modified by Amadori glucose adducts is a plasma-borne factor that activates cell signaling pathways, modulates the expression of growth factors and cytokines, and participates in the pathogenesis of microvascular complications of diabetes. In the present study, streptozotocin diabetic rats were treated with an orally administered compound that inhibits the nonenzymatic glycation of albumin to evaluate whether increased glycated albumin contributes to diabetes-associated abnormalities in the vitreous fluid.

Methods: Vitreous obtained from age-matched nondiabetic and streptozotocin-diabetic rats, half of which received the test compound 2-(3-chlorophenylamino) phenylacetic acid (23CPPA) by oral gavage for 26 weeks, was analyzed by immunoassay for pigment epithelium-derived factor (PEDF), vascular endothelial growth factor (VEGF) and glycated albumin content, by measurement of thiobarbituric acid reactive substances (TBARs) for lipid peroxide products and by colorimetric assay for hyaluronan content.

Vitreous fluid of db/db mice exhibits alterations in angiogenic and metabolic factors consistent with early diabetic retinopathy.

Background: This study evaluated the postulate that the vitreous of diabetic db/db mice, a genetic model of type 2 diabetes that manifests hyperglycemia and insulin resistance, exhibits alterations in angiogenic and metabolic factors that reflect abnormalities in the retinal microvasculature participatory in the pathogenesis of diabetic retinopathy.

Methods: Vitreous obtained from db/db and age-matched nondiabetic db/m mice was analyzed by Western blot for pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF), by immunoassay for type IV collagen, and by measurement of TBARs for lipid peroxide products.

Results: Compared to nondiabetic db/m controls, vitreous from db/db mice contained decreased PEDF and increased VEGF (VEGF:PEDF relative ratio 2.

Inhibiting albumin glycation attenuates dysregulation of VEGFR-1 and collagen IV subchain production and the development of renal insufficiency.

Glomerular cells in culture respond to albumin containing Amadori glucose adducts (the principal serum glycated protein), with activation of protein kinase C-beta(1), increased expression of transforming growth factor (TGF)-beta1, the TGF-beta type II signaling receptor, and the extracellular matrix proteins alpha(1)(IV) collagen and fibronectin and with decreased production of the podocyte protein nephrin. Decreasing the burden of glycated albumin in diabetic db/db mice significantly reduces glomerular overexpression of TGF-beta1 mRNA, restores glomerular nephrin immunofluorescence, and lessens proteinuria, mesangial expansion, renal extracellular matrix protein production, and increased glomerular vascular endothelial growth factor (VEGF) immunostaining. In the present study, db/db mice were treated with a small molecule, designated 23CPPA, that inhibits the nonenzymatic condensation of glucose with the albumin protein to evaluate whether increased glycated albumin influences the production of VEGF receptors (VEGFRs) and type IV collagen subchains and ameliorates the development of renal insufficiency.

Evidence linking glycated albumin to altered glomerular nephrin and VEGF expression, proteinuria, and diabetic nephropathy.

Background: Albumin modified by Amadori-glucose adducts has been linked to the development of diabetic nephropathy through its ability, independent of hyperglycemia, to activate protein kinase C-beta (PKC-beta), up-regulate the transforming growth factor-beta (TGF-beta) system, and stimulate expression of extracellular matrix proteins in glomerular cells, and by the demonstration that reducing the burden of glycated albumin ameliorates renal structural and functional abnormalities in the db/db mouse.

Methods: To probe whether the salutary effects consequent to lowering glycated albumin, which include reduction of albuminuria, relate to an influence of the Amadori-modified protein on nephrin, the podocyte protein critical to regulation of protein excretion, and on the angiogenic vascular endothelial growth factor (VEGF), which induces microvascular permeability, diabetic db/db mice were treated with a small molecule that inhibits the nonenzymatic glycation of albumin.

Results: Compared to nondiabetic db/m mice, diabetic controls exhibited increased urinary excretion of albumin and type IV collagen, elevated renal TGF-beta1 protein levels, reduced glomerular nephrin immunofluorescence and nephrin protein by immunoblotting, and increased glomerular VEGF immunostaining and renal VEGF protein content.

Normalizing glycated albumin reduces increased urinary collagen IV and prevents renal insufficiency in diabetic db/db mice.

Increased excretion of type IV collagen accompanies the accumulation of mesangial matrix, which leads to compromise in the glomerular filtration surface area, during the development of diabetic nephropathy. We postulated that the response of urinary collagen IV would be useful in evaluating possible treatment strategies to arrest the nephropathic process while still at a reversible stage. To test this hypothesis, we examined the effect of a small molecule (22CPPA) that inhibits the formation of glycated albumin, which is causally linked to the pathogenesis of diabetic nephropathy, on collagen IV excretion, albuminuria, and renal function in db/db mice.

Inhibiting albumin glycation in vivo ameliorates glomerular overexpression of TGF-beta1.

Background: Glycated albumin has been causally linked to the pathobiology of diabetic renal disease through its ability to stimulate the expression of transforming growth factor-beta1 (TGF-beta1), activate protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), and promote production of extracellular matrix proteins in cultured glomerular cells. Whether glycated albumin modulates glomerular TGF-beta1 expression in vivo is not known. To address this issue, we assessed glomerular TGF-beta1 expression and pathology in response to reducing the burden of glycated albumin in vivo.