Increased Solubility of Newly Synthesized Collagen in Retinal Capillary Pericyte Cultures by Nonenzymatic Glycosylation

Abstract

The effect of increasing glucose concentration on the solubility of newly synthesized collagen was studied in bovine retinal capillary pericyte (BRCP) cultures. Synchronized pericytes were labeled with [³H]-proline in media containing either 5, 10, 20 or 40 mM glucose, or 5 mM glucose and 100 μg/ml β-aminopropionitrile fumarate (BAPN). The relative rate of collagen synthesis in the medium (soluble collagen) was significantly increased, and the rate in the cell layer (insoluble collagen) was decreased, by an elevated glucose concentration. The increase in collagen solubility with 20 and 40 mM glucose (over normal, 5 mM glucose) was 14 and 42%, respectively, of that obtained with BAPN. BAPN and 4-deoxypyridoxine inhibited BRCP lysyl oxidase activity, but elevated glucose concentrations did not. Increased nonenzymatic glycosylation of newly synthesized collagen was demonstrated in BRCP cultures co-labeled with L-[¹⁴C]-glucose and L-[³H]-proline. The increase in collagen solubility with elevated glucose concentrations therefore appears to be the result of the inhibition of lysyl-derived cross-link formation because of an increased nonenzymatic glycosylation of the ε-amino groups of lysyl residues of newly synthesized collagen in culture.

Inflammation, Aging, and Cancer: Tumoricidal Versus Tumorigenesis of Immunity

“…3). As discussed above, hyperglycemia of diabetes or insulin deficiency-induced generation of ROSs and toxicity to vascular endothelial cells, and/or increased non-enzymatic glycosylation (NEG) of functional proteins (e.g., vascular or extracellular collagen, albumin, hemoglobulin) have been implicated in type-II diabetes complications (e.g., cardiovascular diseases, stroke, retinopathy, nephropathy, neuropathy, hypertension, high serum cholesterol-LDL, or atherosclerosis) [6,18,20,52,82,84,151,190,192,[195][196][197][198][199]. Circulating high glucose levels may induce additional oxidative stress in insulin-independent tissues (e.g., vasculature, retina, RPE, kidney, brain) by modifying or competing with transport, metabolism, and function of important solutes/metabolites or regenerative pathways in ascorbate (vitamin C)-semidehydroascorbate (SDA), pyridoxal phosphate-pyridoxine (vitamin B6), myo-inositol transport and/ or post-modifications of functional proteins, or alterations of NADP ?…”

“…/NADH ratios and mitochondrial respiration (Fig. 3) [195][196][197][198][199]Khatami,personal communications].…”