Structural and functional consequences of increased tubulin glycosylation in diabetes mellitus.

Abstract

The extent ofin vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTPdependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.Diabetes mellitus is often accompanied by the gradual development of diverse and multifocal pathology (1). Insulin and antibiotics have contributed substantially to the management of carbohydrate abnormalities and infection (2). However, deterioration in the microcirculation of retina and kidney and diabetic neuropathy continue to present serious clinical problems (3). It is still unclear whether these complications are uniquely the result of carbohydrate disturbances due to intermittent insulin deficiency or also reflect more subtle genetic or metabolic factors that are, at best, indirectly related to insulin levels (4).Recent data have supported the hypothesis that increased nonenzymatic as well as enzymatic glycosylation ofcellular components, as a result of the elevated glucose concentrations of diabetes, may play a contributory role in diabetic microangiopathy (5), and diabetic cataractogenesis (6). Modification, by the nonenzymatic addition of glucose, has now been reported for a variety of serum, cellular, and interstitial proteins and phospholipids (7-10), suggesting the possibility that unanticipated additional functional changes may yet be observed to result from nonenzymatic (or enzymatic) glycosylation.We present here evidence for in vitro and in vivo glycosylation of brain tubulin as a consequence of exposure to elevated glucose concentrations. The in vitro nonenzymatic glycosylation oftubulin results in the formation ofamorphous aggregates oftubulin and tubulin-associated proteins that remain insoluble in 8 M urea or NaDodSO4. The in vivo formation of glycosylation-induced tubulin aggregates was found to compete with ordered tubulin polymerization in diabetic rats. METHODS AND MATERIALSTubulin Isolation and Measurement. Tubulin was purified from male weanling Sprague-Dawley rat brain according to the procedure of Berkowitz et al. (11). Rats were sacrificed by occipital trauma and cervical dislocation. Brains were removed and weighed and 0.75 ml of purification buffer (11) [50 mM 4-morpholineethanesulfonic acid (Sigma)/2 mM EGTA/1 mM MgSO4/4.0 M glycerol, pH 6.9] was added per gram of brain.Brains were homogenized in a loose-fitting motor-driven Teflon/glass homogeniz...

Receptor for Advanced Glycation End Products (RAGEs) and Experimental Diabetic Neuropathy

“…The increased nonenzymatic glycation of proteins, leading to irreversible formation and deposition of reactive advanced glycation end products (AGEs), may similarly lead to critical abnormalities within the diabetic PNS. AGEs can be detected throughout the central nervous system and PNS in nondiabetic subjects (10). Most importantly, the receptor for AGEs (RAGE) has been demonstrated on hematopoietic cells and endothelial cells, as well as spinal motor neurons and cortical neurons (11,12).…”

Increased protein damage in renal glomeruli, retina, nerve, plasma and urine and its prevention by thiamine and benfotiamine therapy in a rat model of diabetes

“…Increased FL residue content of sciatic nerve and brain of STZ diabetic rats was reported previously using 3 Hlabelled borohydride reduction [25,26]. FL residue content of proteins is influenced by glucose concentration, protein reactivity, repair of FL residues by fructosamine 3-phosphokinase (not available in plasma) [27] and protein half-life.…”

Non-enzymatic glycosylation and the chronic complications of diabetes: an overview

“…One hypothesis is that glycosylation of myelin may yield products which act as signals for recognition and degradation by macrophages, thus inducing excessive myelin turnover and demyelination in diabetes. Increased glycosylation of intracellular tubulins was also observed in diabetic rat brain [51]. Tubulin from diabetic animals exhibited a defect in guanosine triphosphate-induced polymerization, which could be mimicked by glycosylation of normal tubulin in vitro.…”