In this study, we examined the possibility of glycoxidation and lipid peroxidation of low density lipoprotein in atherosclerotic lesions in vivo and in vitro, in order to clarify their role in the pathogenesis of diabetic macroangiopathy. NIDDM patients showed a greater increase in hemoglobin Alc in plasma and carboxymethyllysine (CML), a glycoxidation product, in collagen of an internal thoracic artery than nondiabetic controls. The level of CML significantly correlated with that of hemoglobin Alc in plasma. An immunochemical analysis showed that CML and oxidized phosphatidylcholine (oxPC), one of lipid peroxidation products, were formed in human low density lipoprotein (LDL) which was incubated with copper ion and glucose in vitro. The amount of CML increased in LDL in dose dependent manner of copper ion and glucose added. However, the formation of oxPC depended on the concentration of copper ion, but was independent of the glucose concentration. The formation of CML was completely inhibited either by chelators or by aminoguanidine. On the other hand, the formation of oxPC was completely inhibited by chelators, but partially protected by aminoguanidine. An immunohistochemical analysis demonstrated that CML, oxPC and malondialdehyde (MDA) mainly accumulated in macrophage/foam cells and atheroma, while pyrraline, one of nonoxidative products of glycation, localized in the extracellular matrix in atherosclerotic lesions. These results suggest that the glycoxidation and lipid peroxidation of LDL may synergistically promote the development of atherosclerotic lesions, which may thus contribute to the progression of diabetic macroangiopathy.

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We studied the effect of a high glucose level on gap junctional intercellular communication (GJIC) activity in cultured vascular smooth muscle cells (VSMCs) using the fluorescent dye transfer method. After a 48-h incubation with 22mmol/l glucose (high glucose level), GJIC activity of VSMCs was significantly reduced as compared to the incubation with 5.5 mmol/l glucose (normal glucose level). Treatment of the cells with 12-Otetradecanoylphorbol-13-acetate (TPA;5×10^-8 mol/l), a protein kinase C (PKC) activator, for 1h also significantly reduced GJIC activity. In addition, treatment with calphostin C, a specific PKC inhibitor, for 3h completely restored the glucose-induced reduction of GJIC activity. Western blot analysis showed that connexin 43 (Cx43), which is the major functional protein of GJIC, is present in multiphosphorylated forms:a nonphosphorylated form (P0) and phosphorylated forms (P1, P2, and P3). Incubation of VSMCs with a high glucose level significantly increased the density ratio of P3/P0 as compared to a normal glucose level. Similarly, treatment of the cells with TPA significantly increased the P3/P0 ratio. Then, the increase in P3/P0 density ratio by a high glucose level was restored to the normal range by both staurosporine and calphostin C. These results suggest that high glucose levels may induce the reduction of GJIC activity in cultured VSMCs through excessive phosphorylation of Cx43 mediated by PKC activation. This may contribute to the development of diabetic angiopathy.
Next, we examined the phosphorylation state of Cx43 in the heart of streptozotocin-induced diabetics. The P3/P0 ratio of rat heart was significantly increased in the diabetic rats. To determine whether electrical conduction was affected by excessive phosphorylation of Cx43 in the diabetic rat heart, electrocardiogram was performed. QRS duration was significantly longer in the diabetic rats than their controls. Then, the administration of a PKCβ inhibitor, LY333531, to the diabetic rats completely normalized the prolonged QRS duration. The impaired electrical conduction by decreased gap junction activity may contribute to the pathogenesis of arrythmia and contractile dysfunction shown in diabetics.

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