Altered GAP-43 immunoreactivity in regenerating sciatic nerve of diabetic rats.

Experimental diabetes in the rat is associated with impaired axon regeneration. Successful regeneration depends on the construction of axonal growth cones and establishment of appropriate target connections. The growth-associated protein (GAP)-43 is a major component of the axonal growth cone, and its synthesis and axonal transport are markedly increased during regeneration. The purpose of this study was to determine the effect of experimental diabetes on the synthesis and axonal transport of GAP-43 in regenerating sciatic nerves. Rats were rendered diabetic with 50 mg/kg streptozotocin i.p. Four weeks later, the rats were anesthetized, and one sciatic nerve was crushed to induce regeneration. After 2 weeks, nerves were ligated, and 6 h later, nerve pieces proximal to the ligature and dorsal root ganglia were removed, and proteins were separated by PAGE. Western blots of gels were probed with antibody 10E8/E7 against GAP-43. The presence of GAP-43 was confirmed by immunohistochemistry of nerve sections. Densitometric analysis of the blots showed a 45% reduction in native GAP-43 immunoreactivity in nerve pieces proximal to the ligature (P < 0.05; n = 7). Northern blots of total RNA extracted from pooled dorsal root ganglia were probed with a 32P-radiolabeled cDNA probe for GAP-43. There was no significant difference in the amount of GAP-43 mRNA between diabetic and nondiabetic rats. Immunohistochemistry of sciatic nerve confirmed the reduction in GAP-43 immunoreactivity. We conclude that a defect in turnover or axonal transport of GAP-43 may contribute to the impaired peripheral nerve regeneration in diabetes.

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes.

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.

Co-localization of glucokinase with actin filaments.

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 co-localization of glucokinase with actin filaments was not clearly demonstrated in the pancreatic beta-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 co-localization with actin filaments, may have an important physiological role in metabolic regulation.

Impaired induction of nerve ornithine decarboxylase activity in the streptozotocin-diabetic rat is prevented by the aldose reductase inhibitor ponalrestat.

1. The present study was designed to investigate if the aldose reductase inhibitor ponalrestat is capable of preventing the impairment of the response of ornithine decarboxylase (ODC) to nerve crush in streptozotocin (STZ)-diabetic rats. 2. ODC activity was measured in the dorsal root ganglia of crushed and uncrushed contralateral sciatic nerve of non-diabetic, ponalrestat-treated non-diabetic, STZ-diabetic and ponalrestat-treated STZ-diabetic rats. 3. Twenty four hours after crush, a significant (P less than 0.001) increase in the ratio of ODC activity in ganglia of crushed relative to uncrushed nerves was found in non-diabetic but not in diabetic rats, as expected. In the ponalrestat-treated diabetic rats the ratio was significantly higher (P less than 0.001) than that in the untreated diabetic rats and was not different from that in the non-diabetic group. 4. Ponalrestat also significantly decreased absolute levels of ODC activity in ganglia of uncrushed nerves from diabetic and non-diabetic animals. Despite the near-normal induction of ODC activity by nerve crush in the ponalrestat-treated diabetic animals, absolute ODC activity remained lower than that in ganglia of uncrushed nerves from non-diabetics. 5. We conclude that ponalrestat is able to prevent the impaired induction of ODC in experimental diabetes. The results, however, call into question the relationship between impaired ODC induction and diabetes-induced defects in nerve regeneration, which are insensitive to ponalrestat.

Diabetes alters aromatase enzyme levels in gonadal tissues of rats.

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.

Neurofilament protein phosphorylation in spinal cord of experimentally diabetic rats.

This study was designed to determine if the known decrease in slow axonal transport of proteins in the sciatic nerve of experimentally diabetic rats is related to altered phosphorylation of neurofilament proteins (NFPs). Rats were rendered diabetic with 50 mg/kg of streptozotocin, i.p. At 3 and 6 weeks later, NFPs were prepared from spinal cord. The in vivo phosphorylation state of NFPs was examined by using phosphate-dependent (RT97) and -independent (RMd09) antibodies against high-molecular-mass NFPs on Western blots. Neurofilament-associated kinase activity was also measured in vitro by incubation of NFPs with [32P]ATP. Phosphorylation of all three NFPs (high, medium, and low molecular mass) occurred, as confirmed by gel electrophoresis and autoradiography. At 30 min of incubation, protein-bound radioactivity in NFPs from diabetic animals was reduced to 86.7 +/- 3.4 and 54.3 +/- 19.6% of that in nondiabetic animals at 3 and 6 weeks of diabetes, respectively (p less than 0.001 and p less than 0.05, respectively). NFPs were also incubated with acid phosphatase and rephosphorylated. Results showed that the increased in vivo phosphorylation contributed to the decreased in vitro phosphorylation. Extraction of protein kinases and addition back to the NFPs revealed, in addition, a reduced activity in the diabetic animals of the protein kinases measured in vitro.

Decreased gastro-intestinal responses to salbutamol and serotonin in streptozotocin-induced diabetes: improving effect of insulin in vivo and in vitro.

1. Decreased gastro-intestinal responses to salbutamol (sal) and serotonin (5-HT) in experimental diabetes have been postulated. The present study was designed to investigate whether in vivo and in vitro insulin treatments improve the decreased gastro-intestinal responses. 2. In vivo insulin treatment (166.7 micrograms/kg/day i.p.) for 6 weeks is able to improve both decreased gastro-intestinal beta-adrenergic and serotonergic responses. 3. Insulin incubation in bathing medium for 4-5 hr enhances the decreased gastro-intestinal responses to sal, but not to 5-HT. 4. The above results strongly suggest that the improving effect of insulin on the gastro-intestinal beta-adrenergic responses is direct in nature. In contrast, the improving effect on insulin on the serotonergic responses occurs via an indirect mechanism.

Vanadate treatment reverses gastrointestinal complications in the streptozotocin-diabetic rats.

1. Insulin-like effects of vanadium compounds have been reported in various experimental conditions. Effects of vanadate on the decreased beta-adrenergic responsiveness of the rat duodenum due to streptozotocin diabetes were investigated to determine its influence on diabetic gastro-intestinal complications as well as its effects on the carbohydrate metabolism. 2. Administration of sodium orthovanadate to streptozoticin-diabetic rats in drinking water (0.7 mg/ml) for 4 weeks resulted in an improvement of carbohydrate metabolism noticed by increased serum levels of insulin and decreased blood levels of glucose as reported in previous studies. 3. Vanadate treatment of streptozotocin-diabetic rats also corrected the diabetic changes in the beta-adrenergic responsiveness of the rat duodenum to salbutamol suggesting a beneficial effect on the diabetic complications of rat gastro-intestinal tract. The same treatment with vanadate did not cause any alteration in the beta-adrenergic responsiveness of isolated duodenum from non-diabetic rats. 4. From the findings obtained, it is concluded that vanadate possesses an insulin-like effect on the beta-adrenergic responsiveness of the rat gastro-intestinal tract. Since vanadate treatment did not alter the beta-adrenergic responses of isolated duodenum from non-diabetic rats it seems likely that the insulin-like effect of vanadate is dependent on increased responsiveness of the gastrointestinal tract to circulating insulin.

The effect of vanadyl treatment on vascular responsiveness of streptozotocin-diabetic rats.

Vanadyl sulphate has been demonstrated to possess insulin-like effects in streptozotocin (STZ) diabetic rats, including the normalization of hyperglycaemia and the prevention of diabetes-induced cardiac dysfunction. However, the effectiveness of vanadyl sulphate on diabetes-related vascular aberrations has not been questioned. Hence, in the present work, we have specifically addressed the question of whether chronic oral vanadyl sulphate treatment has any beneficial effect on diabetes-induced changes in vascular reactivity. Male albino rats were injected with a single intravenous dose of STZ (55 mg/kg). Vanadyl sulphate was administered in the drinking water at a concentration of 1 mg/ml from 7 days after the STZ injection and treatment was maintained for 10 weeks. Vanadyl intake was accompanied by decreased blood glucose and serum insulin levels. The effects of diabetes on vascular smooth muscle function were assessed by the responsiveness of aortae to noradrenaline and KCl. Contractile responses of the diabetic aortae were found to be significantly increased as compared with controls. However, there were no significant differences in pD2 values of the agonists in either of the groups. Treatment of diabetic rats with vanadyl sulphate completely prevented the increases in responsiveness of aortae to noradrenaline and KCl. The effect of diabetes on the fast and slow components of noradrenaline-induced contraction was also examined. Both components of the response to noradrenaline were significantly increased in diabetic aortae. These changes were also prevented by vanadyl sulphate treatment. The data demonstrate that 10-week vanadyl sulphate treatment results in improved vascular reactivity of diabetic rats.

Effects of aminoguanidine and N(omega)-nitro-L-arginine methyl ester on vascular hyporeactivity induced by endotoxaemia.

OBJECTIVE: To investigate the effects of endotoxaemia on the reactivity of the aortic bed in rats, and to compare the effects of the nitric oxide (NO) synthase inhibitors aminoguanidine and N(omega)-nitro-L-arginine methyl ester (L-NAME), on endotoxaemia-induced changes in vascular reactivity. DESIGN: Randomised experiment. SETTING: University laboratory, Turkey. SUBJECTS: 54 Wistar rats. INTERVENTIONS: Rats were divided into control (n = 24) and endotoxaemia (n = 30) groups and were treated with an intraperitoneal injection of saline (control) and lipopolysaccharide (20 mg/kg), respectively. Subgroups of control and endotoxaemic rats were given either aminoguanidine or L-NAME by the same route. MAIN OUTCOME MEASURES: Contractile responses to phenylephrine and relaxation responses to acetylcholine 4 hours after treatment. RESULTS: Incubation with aminoguanidine and L-NAME potentiated the phenylephrine-induced contractile response and inhibited acetylcholine-induced relaxation in aortic rings in the control group. The vascular responses to phenylephrine and acetylcholine were less pronounced in the endotoxaemia group, and in vitro incubation with aminoguanidine and L-NAME partially restored the contraction induced by phenylephrine but did not affect the impaired response to acetylcholine. Aminoguanidine given in vivo prevented the impairment of vascular responses to both phenylephrine and acetylcholine whereas L-NAME gave no such protection. CONCLUSION: Aminoguanidine acted similarly to L-NAME when incubated with the tissues in vitro, and did not show selectivity to inducible compared with constitutive isoforms of NO synthase. The finding that aminoguanidine but not L-NAME, prevented the endotoxin-induced impairment of vascular reactivity when administrated in vivo, therefore, suggested a role other than inhibition of NO synthase.

Glycation of brain actin in experimental diabetes.

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.