• Diabetic Membrane Repair Deficiency and Repair Promotion By Vitamin E

      Howard, Amber Cyran; Department of Cellular Biology and Anatomy (5/30/2014)
      Myopathy, characterized by muscle necrosis and atrophy, is a diabetic complication. The myopathy of at least one muscular dystrophy is linked to defective membrane repair. We hypothesized that defective membrane repair is also associated with diabetic myopathy. To test this hypothesis, we monitored repair in intact muscle from diabetic type 1 (INS2Akita+/-) and type 2 (db/db) mouse models. Myocytes were laser injured in the presence of a membrane impermeant dye, and cellular dye uptake through the disruption site was monitored. Dye influx of diabetic myocytes was significantly increased, compared to controls, indicating repair deficiency. This defect was mimicked in cultured cell models by high (30 mM) glucose exposure. Inhibiting the high glucose formation of advanced glycation endproducts (AGE) prevented this repair defect, but was induced in the absence of high glucose exposure by enhanced AGE receptor (RAGE) binding. We conclude that high glucose exposure leads to defective membrane repair in skeletal muscle, and that AGE/RAGE interactions underlie this defect. AGE/RAGE binding also induces generation of reactive oxygen species (ROS), which is increased in diabetes. ROS are also produced in skeletal muscle during eccentric contracts, an act that creates muscle membrane disruptions. Using a potent antioxidant, vitamin E (α-tocopherol), we were able to reverse the high glucose exposure repair defect. Interestingly, diets deficient in vitamin E results in a lethal muscular dystrophy. α-Tocopherol partitions into membrane bilayers where it is thought to act as a membrane stabilizer and/or as an antioxidant. We hypothesize that one important biological role of vitamin E is to promote muscle membrane repair. To test this hypothesis, cultured muscle cells were loaded with α-tocopherol and repair assessed with the laser assay. α-Tocopherol loading significantly decreased cellular dye influx, indicating that repair had been promoted. Strikingly, the HeLa cell, a non-muscle cell that normally displays unrestricted dye influx after laser disruption, e.g. not capable of repair via this form of injury, became repair competent after loading with α-tocopherol. Vitamin C, another antioxidant that can be loaded into cells, also significantly decreased dye influx after laser injury. However, horseradish peroxidase, an antioxidant that lacks transport across the plasma membrane was found to be ineffective in promoting repair. Cells injured in the presence of H2O2, displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If however cells were loaded with vitamin E the H2O2 did not affect repair. We further tested H2O2 exposure in intact mouse skeletal muscle, and found repair to be significantly impaired. However, comparable to vitamin E loading in the cell model, Trolox (a water soluble analog of vitamin E) pretreatment prevented the H2O2 muscle membrane repair defect. We conclude that vitamin E promotes plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.