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Aims: Mitochondrial dysfunction contributes to many forms of peripheral and central nervous system degeneration. Therapies that protect mitochondrial number and function have the potential to impact the progression of conditions such as diabetic neuropathy. We therefore assessed indices of mitochondrial function in dorsal root ganglia (DRG) and brain cortex of the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes and tested the therapeutic impact of a neurogenic compound, NSI-189, on both mitochondrial function and indices of peripheral and central neurological dysfunction. Materials and Methods: ZDF rats were maintained for 16 weeks of untreated diabetes before the start of oral treatment with NSI-189 for an additional 16 weeks. Nerve conduction velocity, sensitivity to tactile and thermal stimuli, and behavioral assays of cognitive function were assessed monthly. AMP-activated protein kinase (AMPK) phosphorylation, mitochondrial protein levels, and respiratory complex activities were assessed in the DRG and brain cortex after 16 weeks of treatment with NSI-189. Results: Treatment with NSI-189 selectively elevated the expression of protein subunits of complexes III and V and activities of respiratory complexes I and IV in the brain cortex, and this was accompanied by amelioration of impaired memory function and plasticity. In the sensory ganglia of ZDF rats, loss of AMPK activity was ameliorated by NSI-189, and this was accompanied by reversal of multiple indices of peripheral neuropathy. Conclusions: Efficacy of NSI-189 against dysfunction of the CNS and PNS function in type 2 diabetic rats was accompanied by improvement of mitochondrial function. NSI-189 exhibited actions at different levels of mitochondrial regulation in central and peripheral tissues.

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Peripheral neuropathy is a major complication associated with diabetes and central neuropathy characterized by Alzheimer's disease-like features in the brain is associated with increased dementia risk for patients with diabetes. Although glucose uptake into the cells of the nervous system is insulin-independent, contribution of impaired insulin support is clearly recognized to play a role, however not yet fully understood, in the development of neuropathy. In this study, we assessed the direct role of insulin on the peripheral nervous system (PNS) and central nervous system (CNS) of insulin-dependent type 1 diabetic rats. Fresh sciatic nerve and hippocampus from control and diabetic rats were incubated with varied ex vivo concentrations of insulin and phosphorylation levels of insulin receptor and glycogen synthase kinase-3 (GSK3ß) were assessed by Western blot analysis. Both the sciatic nerve and hippocampus from type 1 diabetic rats were highly responsive to exogenous insulin with a significantly increased phosphorylation of insulin receptor and GSK3 compared to tissues from control rats. Further, sustained in vivo insulin delivery, not sufficient to restore normal blood glucose, normalized the activation of both insulin receptor and GSK3 in both PNS and CNS tissues. These results suggest that the insulin-signaling pathway is responsive to exogenous insulin in the nervous system of insulin-deficient type 1 diabetic rats and that constant insulin delivery restore normal nerve function and may protect PNS and CNS from damage.

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AIMS: To investigate the efficacy of a pegylated C-peptide (Peg-C-peptide) against indices of peripheral neuropathy in a mouse model of type 1 diabetes and to compare efficacy of this C-peptide analogue against that of the native molecule. METHODS: C57Bl/6 mice were injected with two consecutive doses of streptozotocin (STZ) to induce type 1 diabetes. Mice were treated twice daily with native C-peptide [0.4-1.3 mg/kg subcutaneously (s.c.)] or twice weekly with Peg-C-peptide (0.1-1.3 mg/kg s.c.) for 20 weeks. Motor and sensory nerve conduction velocities, thermal and tactile responses and rate dependent H-wave depression were assessed after 20 weeks of diabetes. Foot skin intraepidermal fibres and corneal nerves were counted, and sciatic nerve substance P and plasma C-peptide levels were also determined. RESULTS: After 5 months of STZ-induced diabetes, mice exhibited significant motor and sensory nerve conduction slowing, thermal hypoalgesia, tactile allodynia and attenuation of rate-dependent depression of the H reflex. These functional disorders were accompanied by nerve substance P depletion but not loss of small sensory fibres in the hind paw epidermis or the cornea. The efficacy of twice-daily treatment with native C-peptide in preventing these disorders was matched or exceeded by twice-weekly treatment with Peg-C-peptide. Both native and Peg-C-peptide also increased corneal nerve occupancy in the sub-basal nerve plexus of control rats. CONCLUSIONS: These data identify actions of C-peptide against novel and clinically pertinent aspects of diabetic neuropathy in mice and also establish Peg-C-peptide as a long-acting therapeutic method of potential clinical value.

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There is an increasing awareness that diabetes has an impact on the CNS and that diabetes is a risk factor for Alzheimer's disease (AD). Links between AD and diabetes point to impaired insulin signaling as a common mechanism leading to defects in the brain. However, diabetes is predominantly characterized by peripheral, rather than central, neuropathy, and despite the common central mechanisms linking AD and diabetes, little is known about the effect of AD on the peripheral nervous system (PNS). In this study, we compared indexes of peripheral neuropathy and investigated insulin signaling in the sciatic nerve of insulin-deficient mice and amyloid precursor protein (APP) overexpressing transgenic mice. Insulin-deficient and APP transgenic mice displayed similar patterns of peripheral neuropathy with decreased motor nerve conduction velocity, thermal hypoalgesia, and loss of tactile sensitivity. Phosphorylation of the insulin receptor and glycogen synthase kinase 3ß (GSK3ß) was similarly affected in insulin-deficient and APP transgenic mice despite significantly different blood glucose and plasma insulin levels, and nerve of both models showed accumulation of Aß-immunoreactive protein. Although diabetes and AD have different primary etiologies, both diseases share many abnormalities in both the brain and the PNS. Our data point to common deficits in the insulin-signaling pathway in both neurodegenerative diseases and support the idea that AD may cause disorders outside the higher CNS.

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AIM: Glucagon-like peptide-1 (GLP-1) is an incretin hormone that induces glucose-dependent insulin secretion and may have neurotrophic properties. Our aim was to identify the presence and activity of GLP-1 receptors (GLP-1Rs) in peripheral nerve and to assess the impact of GLP-1R agonists on diabetes-induced nerve disorders. METHODS: Tissues were collected from streptozotocin-diabetic rats. GLP-1R function was assessed by incubating tissues from normal and diabetic rats with GLP-1R agonists and antagonists and measuring induction of ERK1/2 phosphorylation by Western blot. Streptozotocin-diabetic mice were also treated with the GLP-1R agonist exenatide for 8 weeks to assess the impact of GLP-1R signalling on peripheral nerve function and structure. RESULTS: GLP-1R protein was detected in rat dorsal root ganglia and the neurons and Schwann cells of the sciatic nerve. Protein levels were not affected by streptozotocin-induced diabetes. GLP-1R agonists did not signal via ERK1/2 in sciatic nerve of normal rats. However, GLP-1R agonists significantly increased pERK1/2 levels in sciatic nerves from diabetic rats, indicating that GLP-1Rs are functional in this tissue. Exenatide treatment did not affect blood sugar, insulin levels or paw thermal response latencies in either control or diabetic mice. However, the reductions of motor nerve conduction velocity and paw intraepidermal fibre density seen in diabetic mice were attenuated by exenatide treatment. CONCLUSIONS: These data show that the peripheral nerve of diabetic rodents exhibits functional GLP-1R and suggest that GLP-1R-mediated ERK-signalling in sciatic nerve of diabetic rodents may protect large motor fibre function and small C fibre structure by a mechanism independent of glycaemic control.

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BACKGROUND AND PURPOSE: This study was designed to clarify mechanisms responsible for the anti-allodynic effects of duloxetine in diabetes. EXPERIMENTAL APPROACH: The streptozotocin-induced diabetic rat model was used to compare the efficacy of duloxetine, 5-HT, the 5-HT(2A) receptor agonist [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)] and two antagonists (ketanserin and pruvanserin) on tactile allodynia. KEY RESULTS: Systemic or intrathecal injection of duloxetine alleviated tactile allodynia in diabetic rats. The effect of systemic duloxetine was reduced by intrathecal administration of ketanserin or pruvanserin, indicating participation of spinal 5-HT(2A) receptors in the mechanism of action of duloxetine. In contrast to spinal delivery, systemic and local peripheral injections of ketanserin or pruvanserin alleviated tactile allodynia in diabetic rats. This effect was reversed immediately after systemic or local DOI injection. CONCLUSIONS AND IMPLICATIONS: These results support the involvement of spinal 5-HT(2A) receptors in the ability of duloxetine to ameliorate painful diabetic neuropathy. Our data also suggest that the role of 5-HT(2A) receptors depends on the level of the neuraxis at which activation takes place, with peripheral activation contributing to tactile allodynia in diabetic rats, whereas spinal activation of this receptor alleviates tactile allodynia. The development of selective peripheral 5-HT(2A) receptor antagonists may offer a novel approach for the treatment of diabetic neuropathic pain.

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Recent work suggests that diabetes affects processing of peripheral, spinal and supraspinal signals in the spinal cord. However, there is little evidence for spinal cord lesions that would account for alterations in behavioral responses induced by experimental diabetes. Therefore, we assessed the expression of proteins that might affect neuronal cytoskeletal stability and thus promote dendritic and synaptic reorganization in diabetic rats. Expression of ILK, PINCH, PI3K, GSK-3beta, tau, MAP2, synaptophysin and drebrin in the lumbar spinal cord of non-diabetic and streptozotocin-diabetic rats was assessed by Western-blot analysis and immunocytochemistry after 8 and 20weeks of diabetes. The impact of diabetes on the proteins studied was duration-dependent with changes observed after 20 but not 8weeks of diabetes. ILK and PINCH proteins levels were significantly decreased and both colocalized to neurons and oligodendrocytes. PI3K protein levels were also significantly decreased, while GSK-3beta activity tended to be increased. Phosphorylation of tau and MAP2A/B protein expression were significantly increased, and expression of synaptophysin and drebrin were reduced in diabetic rats. Decreased ILK and PINCH as well as alterations of components of related signaling pathways are associated with tau hyperphosphorylation, MAP2 overexpression and reduction of synaptic proteins in the spinal cord of diabetic rats, suggesting that ILK and PINCH contribute to stabilization of axonal and dendritic structures. However, these changes are not likely the cause of altered behavioral responses in diabetic rats that occur after short-term diabetes, but may contribute to structural changes occurring in long-term diabetes.

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We have evaluated the effect of peripheral insulin deficiency on brain insulin pathway activity in a mouse model of type 1 diabetes, the parallels with Alzheimer's disease (AD), and the effect of treatment with insulin. Nine weeks of insulin-deficient diabetes significantly impaired the learning capacity of mice, significantly reduced insulin-degrading enzyme protein expression, and significantly reduced phosphorylation of the insulin-receptor and AKT. Phosphorylation of glycogen synthase kinase-3 (GSK3) was also significantly decreased, indicating increased GSK3 activity. This evidence of reduced insulin signaling was associated with a concomitant increase in tau phosphorylation and amyloid beta protein levels. Changes in phosphorylation levels of insulin receptor, GSK3, and tau were not observed in the brain of db/db mice, a model of type 2 diabetes, after a similar duration (8 weeks) of diabetes. Treatment with insulin from onset of diabetes partially restored the phosphorylation of insulin receptor and of GSK3, partially reduced the level of phosphorylated tau in the brain, and partially improved learning ability in insulin-deficient diabetic mice. Our data indicate that mice with systemic insulin deficiency display evidence of reduced insulin signaling pathway activity in the brain that is associated with biochemical and behavioral features of AD and that it can be corrected by insulin treatment.

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AIMS/HYPOTHESIS: We investigated spinal and peripheral kappa opioid systems in diabetic rats. MATERIALS AND METHODS: Dynorphin A, N-methyl-D-aspartate (NMDA) and kappa opioid receptor (KOR) were measured in spinal cord, dorsal root ganglia, peripheral nerves and foot skin of control and streptozotocin-induced diabetic rats by immunoassay and Western blotting. Behavioural assessments of paw tactile sensitivity and formalin-evoked hyperalgesia were performed in normal and diabetic rats before and after treatment with asimadoline. RESULTS: Dynorphin A protein levels were significantly increased in peripheral nerves and footpad skin of diabetic rats. Dynorphin A exhibits both anti- and pro-nociceptive properties depending on activation of either KOR or NMDA receptors. Spinal protein levels of these receptors were not changed by diabetes, while KOR levels in the sciatic and peroneal nerves were significantly increased. Exploiting the presence and elevated levels of KOR in the periphery, we investigated the effect of the peripheral KOR agonist asimadoline on formalin-evoked hyperalgesia and tactile allodynia in diabetic rats. Both formalin-evoked hyperalgesia and tactile allodynia in diabetic rats were acutely ameliorated by asimadoline. To confirm that the effect of asimadoline was related to its property as KOR agonist, diabetic rats were pretreated with the selective KOR antagonist nor-binaltorphimine. Intraplantar nor-binaltorphimine abolished the ability of asimadoline to alleviate tactile allodynia in diabetic rats. Systemic and intrathecal nor-binaltorphimine partially inhibited the effect of asimadoline against formalin-evoked hyperalgesia in diabetic rats. CONCLUSIONS/INTERPRETATION: Using selective peripheral KOR agonists to take advantage of elevated peripheral KOR expression may provide a novel therapeutic approach for painful diabetic neuropathy.

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Hydroxyphenylureas are the first main metabolites formed in the environment from pesticide and biocide urea compounds. Because fungi release potent exocellular oxidases, we studied the ability of laccases produced by the white rot fungus, T. versicolor, to catalyze in vitro the transformation of five hydroxyphenylureas, to identify transformation pathways and mechanisms. Our results establish that the pH of the reaction has a strong influence on both the kinetics of the reaction and the nature of the transformation products. Structural characterization by spectroscopic methods (NMR, mass spectrometry) of eleven transformation products shows that laccase oxidizes the substrates to quinones or to polyaromatic oligomers. Slightly acidic conditions favor the formation of quinones as final transformation products. In contrast, at pH 5-6, the quinones further react with the remaining substrate in solution to give hetero-oligomers via carbon-carbon or carbon-oxygen bond formation. A reaction pathway is proposed for each of the identified products. These results demonstrate that fungal laccases could assist the transformation of hydroxyphenylureas.

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Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

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Apolipoprotein E (apoE) whose polymorphic expression is widely associated with Alzheimer's disease (AD) is one of the most studied protein present in cerebral amyloid deposits. Native or fragments of apoE are known to exert neurotoxic effects. We evaluated the effects of apoE oxidation and lipid-association on the viability of human neuroblastoma IMR32 cells. We show that apoE affects cell viability only when it is lipid-associated and applied at a concentration near to that found in plasma, and this whatever the isoform. Oxidized phospholipid-associated apoE has a similar impact on cell viability. These findings show the necessity of including apoE into phospholipids when studying its effect on cell metabolism and underline the probable intervention of surface heparan sulfate proteoglycans (HSPG). It also warrants further studies in order to delineate the pathophysiological importance of apoE.

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Accumulation of oxidized proteins has been demonstrated in the brain of patients suffering from Alzheimer's disease (AD). Among the proteins found in cerebral amyloid deposits, apolipoprotein (apo) E is a polymorphic protein which one specific isoform, apo E4, has been widely associated with AD. Apo E may be linked with AD by its isoform-specific interaction with lipids or other proteins in amyloid plaques. Using the myeloperoxidase oxidative system, we report that oxidation of the three recombinant apo E isoforms is differential (as estimated using immunoblot and high-performance liquid chromatography analysis), with apo E4 being more susceptible than apo E3, which in turn is much more susceptible than apo E2. In addition, susceptibility to thrombin proteolysis is reduced when apo E is oxidized, and oxidation of apo E decreases its incorporation into phospholipid discs by approximately 50%. Oxidation of apo E may contribute to inefficient lipid recycling in the brain, particularly regarding apo E4 and E3. Our results link and strengthen both the E4 allele linkage with AD and the role of protein oxidation in AD. The cerebral mechanisms underlying apo E oxidation and/or myeloperoxidase functions in vivo remain to be assessed.

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The interaction between Bovine Pancreatic Trypsin Inhibitor and thiocyanate was studied using NMR spectroscopy following several experimental approaches. The chemical shift variations of the BPTI protons in the absence and in the presence of increasing thiocyanate concentrations (up to 0.2 M) were significant (> 0.05 ppm) for 30 protein protons belonging to 20 residues. The largest deviation, 0.2 ppm, was observed for the amide backbone proton of Arg42 in the absence of thiocyanate and in the presence of 40 molar equivalents of thiocyanate. The influence of the presence of thiocyanate on the electrostatic potential surrounding the protein was demonstrated by NOESY spectra selective at the water frequency: the presence of SCN- favours acid catalysed exchange and disfavours base catalysis. However, a specific effect of thiocyanate was pointed out since the comparison of the chemical shifts in the presence of 40 molar equivalents of KSCN and KCl, respectively, showed much more as well as larger deviations compared to measurements in the absence of salt. A dissociation constant, KD, for a 1/1 complex between BPTI and thiocyanate was calculated from chemical shifts measurements: KD = 89 +/- 8 mM. A second value, KD = 99 +/- 10 mM, was extracted from SC15N relaxation time measurements.

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The quantitative measurement of the salt content in solid protein samples was performed using X-ray fluorescence. Linear calibration curves were obtained for chloride, calcium and sulfur using sulfur and chloride as internal standards in the range 1-10 protein molar equivalents. The detection limit was approximately 0.02 molar equivalents for chloride and less than 0.01 molar equivalents for calcium. X-ray fluorescence thus provides a non-destructive sensitive method of testing the efficiency of different purification methods. Commercial hen egg white lysozyme samples contain from 15 to 46 molar equivalents of chloride, whereas the calcium content remains less than 0.2 equivalents. Deionization on ion-exchange resins is a very efficient tool for removing ionic species since deionized lysozyme samples contain less than 0.34 molar equivalents of chloride. Extensive dialysis against water only partially removes chloride ions, the residual chloride content corresponding to the number of counter-ions necessary to ensure the electroneutrality of lysozyme when dissolved in water.

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Apolipoprotein E, the most common apolipoprotein found in the brain, is linked to several pathologies like Alzheimer's disease. Apolipoprotein E directly binds to beta-amyloid with a strong affinity. Myeloperoxidase, a protein secreted by neutrophils and involved in the inflammatory process, is also present in the brain. In vitro myeloperoxidase oxidation of recombinant human apolipoprotein E leads to fragmentation of the protein with low concentrations of hydrogen peroxide and polymerization with higher concentrations. Comparison with bovine serum albumin shows a higher susceptibility of apolipoprotein E to myeloperoxidase oxidation, which may have importance in the Alzheimer's disease process.

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The precipitation equilibria of hen egg-white lysozyme and bovine serum albumin from aqueous solutions by caprylic acid were studied. A thermodynamic equilibrium was obtained, and the compositions of both the precipitate and the aqueous phases were measured to establish phase diagrams for both systems. The precipitate was not pure protein, but also contained large amounts of water and caprylic acid. At constant initial pH and ionic strength, the composition of both phases depended only on the overall composition of the system. This suggests that protein precipitation by fatty acids should be pictured as liquid-liquid rather than solid-liquid equilibria. The precipitated proteins retained high biological activity. (c) 1996 John Wiley & Sons, Inc.

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CYP2D, a genetically variable isoform of cytochrome P450, has been characterized mainly in the liver and the brain of mammals by measurement of debrisoquine hydroxylase activity. Moreover, 'poor debrisoquine metabolizer' phenotype is significantly increased in Parkinson's disease patients. We present here the first demonstration that the activity of the CYP2D isoform can be characterized in rat brain microsomes by the measurement of dextromethorphan O-demethylase capacity. The cerebral formation of dextrorphan, an antagonist of the N-methyl-D-aspartate receptor, was inhibited by the presence of quinidine and N-methyl-4-phenylpyridinium (MPP+), a dopaminergic neurotoxin inducing a chemical parkinsonism in humans.

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The partition of cytochrome c between an aqueous phase in equilibrium with a microemulsion composed of sodium dodecylbenzenesulfonate, butanol, and decane was studied. By adjusting the pH and the ionic strength in the aqueous phase, cytochrome c can be quantitatively extracted in the microemulsion as long as the protein concentration in the organic phase is less than the initial concentration of micelles. Above this limit, an intermediate phase, containing part surfactant and part cytochrome c, is located between the aqueous and microemulsion phases. The role of electrostatic interactions in the protein partitioning is shown by varying the composition of the phases (pH, salinity, surfactant, and cosurfactant concentrations) and then discussed. Extraction depends on the relative values of the pH of the aqueous phase and the pI of cytochrome c, the size of the micelles, and the protonation of acidic residues. Cytochrome c can be recovered in an aqueous phase by adding butanol in the microemulsion phase. In such conditions, cytochrome c retains 90% of its initial activity, as measured before the extraction step.

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