Ganglioside Treatment Modifies Abnormal Elemental Composition in Peripheral Nerve Myelinated Axons of Experimentally Diabetic Rats

Abstract: Effects of ganglioside administration on elemental composition of peripheral nerve myelinated axons and Schwann cells were determined in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats (50 days after administration of streptozocin) exhibited a loss of axoplasmic K and Cl concentrations in sciatic nerve relative to control, whereas intraaxonal levels of these elements increased in tibial nerve. These regional changes in diabetic rat constitute a reversal of the decreasing proximodistal gradients for K and Cl concentrations that characterize normal peripheral nerve. Treatment of diabetic rats with a ganglioside mixture for 30 days (initiated 20 days after the administration of streptozocin) returned proximal sciatic nerve axoplasmic K and Cl concentrations to control levels, whereas in tibial axons, concentrations of these elements increased further relative to diabetic levels. Also in the ganglioside/diabetic group, mean axoplasmic Na concentrations were reduced and Ca levels were elevated. Mixed ganglioside treatment of nondiabetic rats significantly increased axoplasmic dry weight concentrations of K and Cl in proximal sciatic and tibial axons. Schwann cells did not exhibit consistent alterations in elemental content regardless of treatment group. Changes in elemental composition evoked by ganglioside treatment of diabetic rats might reflect the ability of these substances to stimulate Na⁺,K⁺-ATPase activity and might be related to the mechanism by which gangliosides improve functional deficits in experimental diabetic neuropathy.     

Variability in the Structural Requirements for Binding of Human Monoclonal Anti-Myelin-Associated Glycoprotein Immunoglobulin M Antibodies and HNK-1 to Sphingoglycolipid Antigens

A high proportion of patients with neuropathy have immunoglobulin M (IgM) paraproteins that react with carbohydrate determinants on the myelin-associated glycoprotein (MAG) and two sphingoglycolipids, 3-sulfoglucuronyl paragloboside (SGPG) and 3-sulfoglucuronyl lactosaminyl paragloboside. In order to characterize the fine specificities of these human antibodies further, the binding of 10 anti-MAG paraproteins to several chemically modified derivatives of SGPG was compared with the binding to intact SGPG by both TLC-overlay and enzyme-linked immunosorbent assay. The following derivatives were tested: the desulfated lipid, glucuronyl paragloboside (GPG); the methyl ester of GPG (MeGPG); the methyl ester of SGPG, 3-sulfomethylglucuronyl paragloboside (SMeGPG); and 3-sulfoglucosyl paragloboside (SGlcPG) produced by reduction of the carboxyl group of the glucuronic acid with sodium borohydride. All 10 IgM paraproteins and the related mouse IgM antibody, HNK-1, reacted most strongly with intact SGPG, but variations in the reactivity with the derivatives revealed striking differences in the structural requirements for binding between the antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species Distribution of Paraoxon-Resistant Brain Polypeptides Radiolabeled with Diisopropyl Phosphorofluoridate ([3H]DiPF): Electrophoretic Assay for the Aged Polypeptide of [3H]DiPF-Labelled Neuropathy Target Esterase

Brain neuropathy target esterase is identified as a paraoxon-resistant, mipafox-sensitive esterase that can be labelled with [3H]diisopropyl phosphorofluoridate. During "aging" of the labelled (inhibited) esterase, half the label (one isopropyl group) is transferred to a site (of the same molecular weight in sodium dodecyl sulphate) whence it may be released in volatile form by treatment with alkali. Our previously published procedure for complete extraction in a form suitable for scintillation counting of tritium-labelled proteins from polyacrylamide gels includes treatment of part-solubilised gels with alkali. Particles from brain of the hen, pig, sheep, guinea-pig, and rat were preincubated with paraoxon with or without mipafox, treated with [3H]diisopropyl phosphorofluoridate, and solubilised in sodium dodecyl sulphate. Labelled polypeptides (except from the rat) were separated by electrophoresis. Both mipafox-sensitive labelling and "volatilisable counts" were located principally in the 155-kilodalton region, with the residues dispersed throughout the gels. The quantities of paraoxon-resistant, mipafox-sensitive labelling sites and of "volatilisable counts" (in pmol/particles from 1 g) were, respectively, 12.2 and 8.65 in hen brain, 9.80 and 6.82 in pig, 8.48 and 5.46 in sheep, 4.46 and 4.01 in guinea-pig, and 4.91 and 2.08 in rat. The "volatilisable count" assay seems more specific for neuropathy target esterase and is easier and more precise than assays based on differences in labelling of two samples, each subjected to much processing. Hydrolytic activity of particles taken before labelling was measured against phenyl valerate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of Na,K-ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n-3 Fatty Acids

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase α subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1- and α3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.     

Impact of diabetic polyneuropathy and cardiovascular autonomic neuropathy on the excretion of urinary 8-epi-PGF2α and its metabolites (2, 3-dinor and 2, 3-dinor-5, 6-dihydro)

The objective of this study was to establish if diabetes in the presence of polyneuropathy (PN) and/or cardiovascular autonomic neuropathy (CAN) is associated with alterations in the amounts of 8-epi-PGF_2α (IP) and its metabolites including 2, 3-dinor-8-epi-PGF_2α (dinor-IP) and 2, 3-dinor-5, 6 dihydro-8-epi-PGF_2α (dinor-dihydro-IP) in urine. Mass spectrometric separation showed that excretion of IP was similar in the PN+/CAN ? and PN+/CAN+ groups but higher than in the PN ? /CAN ? group (n = 103, 22 and 60, respectively; P < 0.05). By contrast, excretion of dinor-IP or dinor-dihydro-IP were similar in the PN ? /CAN ? and PN+/CAN ? groups but higher than in PN+/CAN+ group. Correlations were obtained between IP and dinor-IP or dinor-dihydro-IP (r = 0.30; P < 0.001 and r = 0.31; P < 0.001, respectively). A significant association was also observed between dinor-IP and dinor-dihydro-IP (r = 0.48; P < 0.001). In conclusion, these biomarkers should prove useful in studies evaluating the impact of therapeutic drugs or antioxidant interventions aimed at delaying the onset of diabetic complications.     

Aldose Reductase Inhibitors

Aldose reductase ([EC1.1.1.21]: AR) acts on the first step of the polyol metabolic pathway to catalyze the reduction of glucose to sorbitol with NADPH as a coenzyme. Hyperactivity of the pathway in individuals with high blood glucose level is closely related to the onset or progression of diabetic complications. AR inhibitors have therefore been noted as possible pharmacotherapeutic agents for the treatment of diabetic complications. One AR inhibitor has been on the market in Japan, while some potent inhibitors are in clinical trials. Reviewed are the physiological roles of AR, the chemical structures of AR inhibitors, interactions of AR inhibitors with AR using X-ray studies, and the following potencies of AR inhibitors: in vitro activities for AR, in vitro selectivities between AR and aldehyde reductase, their pharmacological effects in vivo, and their effectiveness in clinical trials. Also discussed are directions for the design of future AR inhibitors.     

Axonopathy-Inducing 1,2-Diacetylbenzene Forms Adducts with Motor and Cytoskeletal Proteins Required for Axonal Transport

The aromatic hydrocarbon 1,2-diacetylbenzene (1,2-DAB) is a protein-reactive γ-diketone metabolite of the neurotoxic solvent 1,2-diethylbenzene (1,2-DEB). The effect of neurotoxic 1,2-DAB and its non-neurotoxic isomer 1,3-DAB has been studied on motor proteins and cytoskeletal proteins of rat spinal cord (SC). For in vitro studies, SC slices were incubated with 1, 2, 5, 10 mM of DAB isomers for 30 min at 37°C. For in vivo studies, rats received (i.p.) 20 mg/kg/day of 1,2-DAB or 1,3-DAB, or vehicle (2% acetone in saline), 5 days a week for 2 weeks. Spinal cord and sciatic nerve proteins were subjected to Western blotting using monoclonal mouse antibodies to NF-M, kinesin, dynein, and tau. Proteins were quantified and paired mean comparisons performed to assess concentration-dependent changes in native protein bands. In vitro, 1,2-DAB produced a concentration-dependent decrease of motor and cytoskeletal proteins. While dynein and tau appeared similarly affected by 1,2-DAB, kinesin was most affected by the toxicant. In vivo, 1,2-DAB affected motor and cytoskeletal proteins of sciatic nerves and spinal cord differentially. In general, sciatic nerve proteins were much more affected than spinal cord proteins. The results show that motor proteins that drive axonal transport anterogradely (kinesin) and retrogradely (dynein), cytoskeletal protein NF-M, which is slowly transported in the anterograde direction, and microtubule-associated protein, tau, which is involved in axonal transport, are differentially impacted by 1,2-DAB. By contrast, non-neurotoxic isomer 1,3-diacetylbenzene (1,3-DAB), had no adverse effect on neural proteins either in vitro or in vivo. 2D-Differential in gel electrophoresis (2D-DIGE) of sciatic nerves from neurotoxic 1,2-DAB and non-neurotoxic 1,3-DAB treated rats revealed 197 and 304 protein spots, respectively. This paper is dedicated to my long-time friend Naren L. Banik, Ph.D.     

Calcium signalling in sensory neurones and peripheral glia in the context of diabetic neuropathies

Peripheral sensory nervous system is comprised of neurones with their axons and neuroglia that includes satellite glial cells in sensory ganglia, myelinating, non-myelinating and perisynaptic Schwann cells. Pathogenesis of peripheral diabetic polyneuropathies is associated with aberrant function of both neurones and glia. Deregulated Ca²⁺ homoeostasis and aberrant Ca²⁺ signalling in neuronal and glial elements contributes to many forms of neuropathology and is fundamental to neurodegenerative diseases. In diabetes both neurones and glia experience metabolic stress and mitochondrial dysfunction which lead to deregulation of Ca²⁺ homeostasis and Ca²⁺ signalling, which in their turn lead to pathological cellular reactions contributing to development of diabetic neuropathies. Molecular cascades responsible for Ca²⁺ homeostasis and signalling, therefore, can be regarded as potential therapeutic targets.