Clyde W. Shearman

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. RESULTS Compared with that of nondiabetic controls, vitreous of diabetic rats contained decreased PEDF, increased VEGF, higher VEGF/ PEDF ratio, and elevated levels of TBARs, glycated albumin, and hyaluronan. These changes were significantly attenuated in rats treated with test compound despite the presence of marked hyperglycemia. CONCLUSIONS Results indicate that inhibiting the formation of glycated albumin, which is increased in diabetes, ameliorates vitreous changes in angiogenic and metabolic factors associated with the development of diabetic retinopathy. The observed improvement in vitreous alterations associated with reductions in glycated albumin suggests that elevated levels of glycated albumin play a retinopathogenic role in diabetes that is operative and that can be therapeutically addressed independently of glycemic status.

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 2.2 ± 0.3 and 1.0 ± 0.1 in db/db vs. db/m, respectively; p < 0.05), and elevated concentrations of lipid peroxide products (187 ± 43 and 84 ± 15 ng/ml in db/db vs. db/m, respectively; p < 0.05) and type IV collagen (5.2 ± 0.7 and 3.1 ± 0.4 nmol/ml in db/db vs. db/m, respectively; p < 0.05). These changes were observed at age 18–20 weeks, consistent with an early stage in the development of retinal microvascular pathology. Conclusions: The findings support the potential usefulness of vitreous from the db/db mouse as a model tissue for investigation of pathogenetic factors and assessment of therapeutic interventions in early diabetic retinopathy.

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. The findings indicate that fatty acids impede the ability of albumin to undergo Amadori glucose modification and induce conformational changes affecting exogenous ligand binding, and that nonenzymatic glycation of albumin induces alterations in structural and functional properties that may have import in lipid transport and atherogenesis.

Inhibiting Amadori-modified albumin formation improves biomarkers of podocyte damage in diabetic rats

Recent studies have shown that urinary excretion of podocyte proteins is an indicator of podocyte injury, and that podocyte abnormalities and elevated concentrations of Amadori‐modified glycated albumin (AGA) are linked to the development of diabetic nephropathy and to each other. We evaluated relationships between urinary markers of podocyte damage, increased AGA and filtration function in rats made diabetic by streptozotocin injection and treated for 8 weeks with a compound that inhibits the formation of AGA, with age‐matched nondiabetic and diabetic rats serving as controls. Blood and urine were collected for measurement of glycated albumin, creatinine, albumin, nephrin, podocalyxin, and βig‐h3 protein. The elevated circulating concentrations of glycated albumin and higher urinary levels of these podocyte markers as well as of albumin that were observed in diabetic rats compared with nondiabetic controls were significantly reduced in animals receiving test compound, and decrease in urinary biomarkers correlated with reduction in AGA. The results provide evidence that lowering the concentration of AGA, independent of filtration status and hyperglycemia, reduces urinary nephrin, podocalyxin, and βig‐h3 protein, linking the increased glycated albumin associated with diabetes to podocyte abnormalities and shedding of podocyte proteins into the urine.

Reduction of Aβ42 in brains of transgenic APPswe mice by 2-3-chlorophenylaminophenylacetate

1 Imbalanced generation of the Aβ42 peptide from the amyloid β protein precursor (APP) is implicated in the pathogenesis of Alzheimers disease. 2 The present study is the first to evaluate the ability of 2‐[3‐chlorophenylamino]phenylacetic acid (GLY‐230), a new drug in clinical development for the treatment of vascular complications of diabetes, to modulate Aβ42 levels in transgenic mice expressing APP. 3 Oral administration of 7.5 mg/kg GLY‐230 twice a day for 14 days to APPswe transgenic mice aged 3 months significantly reduced brain Aβ42 and increased plasma Aβ42 levels by 50 and 20%, respectively. 4 GLY‐230 readily entered the brain after administration of a dose (7.5 mg/kg) that decreased brain Aβ42. 5 These results are the first to demonstrate that GLY‐230, which exhibits antiglycation but no cyclo‐oxygenase inhibitory properties, lowers brain Aβ42 levels in this experimental model of Alzheimers disease.