Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with 3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of 32P into purified neurofilament proteins when they were incubated with 32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.

A portion of glucokinase appeared to be co-localized with actin filaments in the cytoplasm of cultured rat hepatocytes incubated with 25 mM glucose. When liver-or islet-type glucokinase was transiently expressed in COS-7 cells, the expressed glucokinase was also co-localized with actin filaments in the cytoplasm of these transfected cells. Although colocalization of glucokinase with actin filaments was not clearly demonstrated in the pancreatic P-cell line MIN6, islet glucokinase was found to be present in both the nucleus and the cytoplasm, though predominantly in the nucleus. These findings suggest that subcellular localization of glucokinase, including colocalization with actin filaments, may have an important physiological role in metabolic regulation.© 1997 Federation of European Biochemical Societies.

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Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two‐dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose‐6‐phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G‐actin to form F‐actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet‐derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.

Diabetes mellitus (DM) is associated with increased risk of reproductive problems. Estrogens have important roles in reproductive processes in both genders. Aromatase catalyzes the conversion of androgens to estrogens and is expressed in a variety of tissues. Although it is known that insulin regulate the activity of aromatase, there are few data about the effects of diabetes on this enzyme. The aim of the present study was to investigate the effects of experimental diabetes on aromatase expression levels in ovary, testis, uterus, and vas deferens tissues of female and male rats. Rats were injected with streptozotocin to induce diabetes. At the end of 4 and 12 weeks, tissue homogenates were prepared and evaluated for aromatase proteins by western blot. Uterus and vas deferens smooth muscle responses were also evaluated. Aromatase expression levels in ovary were significantly decreased both in 4 and 12 weeks of diabetes. In testis, enzyme levels were not altered at 4 weeks, but significantly decreased at 12 weeks of diabetes. In uterus and vas deferens tissues, no significant differences were observed at aromatase immunoreactivity but uterus and vas deferens smooth muscle responses were altered. These results indicated for the first time that DM altered the expression levels of aromatase both in ovary and testis but did not affect enzyme levels in uterus and vas deferens tissues. Altered smooth muscle responses did not correlate with tissue aromatase levels. Altogether, these findings lead us to suggest that aromatase might be an important target molecule in sexual dysfunction seen in DM.