Abstract
Atherosclerosis is the main cause of morbidity and mortality in diabetes, yet the underlying mechanisms remain unclear. Retention of atherogenic lipoproteins by vascular proteoglycans is thought to play a key role in the development of atherosclerotic lesions. High glucose levels cause a variety of diabetic complications by several mechanisms, including upregulation of the hexosamine pathway. Glucosamine, a component of the hexosamine pathway, is a precursor for the synthesis of glycosaminoglycan components of proteoglycans. This study evaluated whether high glucose or glucosamine supplementation of vascular smooth muscle cells would increase proteoglycan synthesis, leading to increased lipoprotein retention. Aortic smooth muscle cells were exposed to physiologic (5.6 mM) or high (25 mM) glucose levels, such as seen in diabetes, or to glucosamine (12 mM). Extracellular proteoglycans were characterized by sulfate incorporation, molecular sieve chromatography, and SDS-PAGE. LDL interactions were assessed by affinity chromatography and gel mobility shift assay. Proteoglycans synthesized in the presence of high glucose demonstrated no differences in size, sulfate incorporation, or LDL binding affinity compared with proteoglycans synthesized under physiological glucose conditions. However, proteoglycans synthesized in the presence of glucosamine had smaller glycosaminoglycan chains than control proteoglycans with a corresponding decrease in lipoprotein retention.Thus, glucose and glucosamine have different effects on proteoglycan biosynthesis and different effects on lipoprotein retention.
Highlights
Atherosclerosis is the main cause of morbidity and mortality in diabetes, yet the underlying mechanisms remain unclear
Despite the finding of cytotoxicity with azaserine, proteoglycans synthesized in the presence of azaserine were not different from those synthesized under control conditions, suggesting that azaserine did not have a direct effect on proteoglycan synthesis
To examine the effect of glucosamine supplementation on the glycosaminoglycan components of the proteoglycans, proteoglycans synthesized in the presence of glucosamine (12 mM) or synthesized under control conditions were treated with sodium borohydride to release their glycosaminoglycan chains
Summary
Arterial smooth muscle cells isolated from monkey ( Macaca nemestrina) thoracic aortas were plated at a density of 2.5 ϫ 105 cells per 60-mm dish and grown to confluence in commercially available DMEM with 5.6 mM glucose, supplemented with pyruvate, nonessential amino acids, penicillin (10 5 U/l), streptomycin (105 g/l), glutamine, and 5% calf serum. Quiescent cells were exposed to high (25 mM) or physiologic (5.6 mM) concentrations of glucose for 24 h in the presence of 5% serum. To test the role of the hexosamine pathway, quiescent cells were exposed to high glucose (25 mM) DMEM, or to high glucose medium in the presence of the GFAT inhibitor azaserine (5 M) [14]. Cells were exposed to these conditions for 24 h in the presence of 5% serum, and were metabolically labeled under these conditions, as described earlier. Time course studies were performed by exposing quiescent cells to physiologic glucose (5.6 mM) or various concentrations of glucosamine (0.12, 1.2, or 12 mM) for 8, 16, or 24 h in the presence of 35SO4 for the last 8 h of incubation
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