In vivo expression of GLP-1/IgG-Fc fusion protein enhances beta-cell mass and protects against streptozotocin-induced diabetes.

Glucagon-like peptide 1 (GLP-1) and its analogue exendin-4 (Ex4) have displayed potent glucose homeostasis-modulating characteristics in type 2 diabetes (T2D). However, there are few reports of effectiveness in type 1 diabetes (T1D) therapy, where there is massive loss of beta cells. We previously described a novel GLP-1 analogue consisting of the fusion of active GLP-1 and IgG heavy chain constant regions (GLP-1/IgG-Fc), and showed that in vivo expression of the protein, via electroporation-enhanced intramuscular plasmid-based gene transfer, normalized blood glucose levels in T2D-prone db/db mice. In the present study, GLP-1/IgG-Fc and Ex4/IgG-Fc were independently tested in multiple low-dose streptozotocin-induced T1D. Both GLP-1/IgG-Fc and Ex4/IgG-Fc effectively reduced fed blood glucose levels in treated mice and ameliorated diabetes symptoms, where as control IgG-Fc had no effect. Treatment with GLP-1/IgG-Fc or Ex4/IgG-Fc improved glucose tolerance and increased circulating insulin and GLP-1 levels. It also significantly enhanced islet beta-cell mass, which is likely a major factor in the amelioration of diabetes. This suggests that GLP-1/IgG-Fc gene therapy may be applicable to diseases where there is either acute or chronic beta-cell injury.

Gene therapy of diabetes using a novel GLP-1/IgG1-Fc fusion construct normalizes glucose levels in db/db mice.

Glucagon-like peptide (GLP-1), a major physiological incretin, plays numerous important roles in modulating blood glucose homeostasis and has been proposed for the treatment of type 2 diabetes. The major obstacles for using native GLP-1 as a therapeutic agent are that it must be delivered by a parenteral route and has a short half-life. In an attempt to develop a strategy to prolong the physiological t(1/2) and enhance the potency of GLP-1, a fusion protein consisting of active human GLP-1 and mouse IgG(1) heavy chain constant regions (GLP-1/Fc) was generated. A plasmid encoding an IgK leader peptide-driven secretable fusion protein of the active GLP-1 and IgG(1)-Fc was constructed for mammalian expression. This plasmid allows for expression of bivalent GLP-1 peptide ligands as a result of IgG-Fc homodimerization. In vitro studies employing purified GLP-1/Fc indicate that the fusion protein is functional and elevates cAMP levels in insulin-secreting INS-1 cells. In addition, it stimulates insulin secretion in a glucose concentration-dependent manner. Intramuscular gene transfer of the plasmid in db/db mice demonstrated that expression of the GLP-1/Fc peptide normalizes glucose tolerance by enhancing insulin secretion and suppressing glucagon release. This strategy of using a bivalent GLP-1/Fc fusion protein as a therapeutic agent is a novel approach for the treatment of diabetes.

Intracellular lithium and cyclic AMP levels are mutually regulated in neuronal cells.

In this work, we studied the effect of intracellular 3',5'-cyclic adenosine monophosphate (cAMP) on Li+ transport in SH-SY5Y cells. The cells were stimulated with forskolin, an adenylate cyclase activator, or with the cAMP analogue, dibutyryl-cAMP. It was observed that under forskolin stimulation both the Li+ influx rate constant and the Li+ accumulation in these cells were increased. Dibutyryl-cAMP also increased Li+ uptake and identical results were obtained with cortical and hippocampal neurons. The inhibitor of the Na+/Ca2+ exchanger, KB-R7943, reduced the influx of Li+ under resting conditions, and completely inhibited the effect of forskolin on the accumulation of the cation. Intracellular Ca2+ chelation, or inhibition of N-type voltage-sensitive Ca2+ channels, or inhibition of cAMP-dependent protein kinase (PKA) also abolished the effect of forskolin on Li+ uptake. The involvement of Ca2+ on forskolin-induced Li+ uptake was confirmed by intracellular free Ca2+ measurements using fluorescence spectroscopy. Exposure of SH-SY5Y cells to 1 mm Li+ for 24 h increased basal cAMP levels, but preincubation with Li+, at the same concentration, decreased cAMP production in response to forskolin. To summarize, these results demonstrate that intracellular cAMP levels regulate the uptake of Li+ in a Ca(2+)-dependent manner, and indicate that Li+ plays an important role in the homeostasis of this second messenger in neuronal cells.

Mechanism of inhibition of mitochondrial respiratory complex I by 6-hydroxydopamine and its prevention by desferrioxamine.

Inhibition of mitochondrial complex I by 6-hydroxydopamine was studied in brain and liver preparations. NADH-quinone reductase activity of this complex from rat brain was inhibited by 6-hydroxydopamine partially uncompetitively with respect to NADH with a value of Ki 0.051 +/- 0.014 mM. The inhibition patterns for liver NADH-quinone reductase were more complicated than those obtained with the brain enzyme. Desferrioxamine behaved as a 'competitive' activator of complex I from both liver and brain (Ka = 2 mM and 0.02 mM, respectively). It also protected brain complex I against the inhibition by increasing Ki value about 10-fold. Furthermore, in the presence of desferrioxamine the residual activity of enzyme-substrate-inhibitor complex was increased. The data suggest that desferrioxamine does not compete directly with 6-hydroxydopamine for binding to the inhibitory site, but induces a conformation which is unfavorable for the binding of the inhibitor to the protein. The qualitative and quantitative differences between the behavior of the liver and brain enzyme complexes indicate that the assumption that the behavior of liver mitochondria can be used as a model for the situation in brain should be reconsidered.

Mechanism of 6-hydroxydopamine neurotoxicity.

The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) has recently been found to be formed endogenously in patients suffering from Parkinson's disease. In this article, we highlight the latest findings on the biochemical mechanism of 6-OHDA toxicity. 6-OHDA has two ways of action: it easily forms free radicals and it is a potent inhibitor of the mitochondrial respiratory chain complexes I and IV. The inhibition of respiratory enzymes by 6-OHDA is reversible and insensitive towards radical scavengers and iron chelators with the exception of desferrioxamine. We conclude that free radicals are not involved in the interaction between 6-OHDA and the respiratory chain and that the two mechanisms are biochemically independent, although they may act synergistically in vivo.

Apomorphine is a highly potent free radical scavenger in rat brain mitochondrial fraction.

Ergoline-derived dopamine receptor agonists, like pergolide or bromocryptine, have recently attracted attention as potential neuroprotective drugs. The classical mixed type dopamine D1 and D2 receptor agonist apomorphine, although used clinically in the therapy of Parkinson's disease, has never been examined for any properties related to neuroprotection. In this paper, we examine the effects of 0.1-100 microM apomorphine on ascorbate/iron-stimulated free radical processes in rat brain mitchondrial fraction. Lipid peroxidation as assayed by the thiobarbituric acid reaction can be completely inhibited by submicromolar concentrations of apomorphine (0.3 microM with 2.5 microM Fe2+ and 0.6 microM with 5.0 microM Fe2+), which proved to be more than twice as effective as desferrioxamine and twenty times as compared with dopamine. The inhibition of lipid peroxidation in mitochondria correlates with an increased rate of apomorphine oxidation. The formation of protein carbonyls, which is generally less sensitive to antioxidants, could be significantly reduced by apomorphine. In the model system we employed, apomorphine was more active than dopamine, desferrioxamine, or pergolide in preventing the formation of thiobarbituric reactive substances. The time course of the reaction suggests that apomorphine acts as a radical scavenger and that its iron chelating properties may not be of major importance. Since oxidative stress has been implicated in Parkinson's disease, the role of apomorphine as a neuroprotective is worthy of examination.

Luminescence investigations of the adsorption properties of disperse Al(2)O(3) surfaces by means of selective laser excitation.

A new method for the determination of the adsorption activity of disperse materials by means of inorganic luminescent probes under selective laser excitation has been developed and tested for the investigation of the adsorption properties of disperse Al(2)O(3) surfaces. Water-uranyl complexes have been used as luminescent probes for these experiments, and the photoluminescence spectra of UO(2)(2+) molecular ions adsorbed on the disperse Al(2)O(3) surfaces were investigated. The luminescence properties of this adsorption system, as in the previously studied case of SiO(2), were determined by the type and structure of the adsorption complexes (AC) formed. Different ACs cause many (7 observed) "elementary" luminescence spectra. The water-uranyl-complex adsorption binding energies were obtained. The values of the binding energies and the electric field strength of the surface active centers support the validity of the water-uranyl model of AC and the electrostatic consideration used.

Nature of inhibition of mitochondrial respiratory complex I by 6-Hydroxydopamine.

The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron (III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.

Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine.

The enzymes of mitochondrial respiratory chain, NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV), were completely inhibited by 6-hydroxydopamine with IC50 = 10.5 microM and IC50 = 34 microM respectively. The enzyme inhibition was insensitive to the change of NADH or cytochrome c concentrations. The extent of complex I inhibition decreased as a consequence of both non-enzymatic and monoamine oxidase-catalyzed oxidation of 6-hydroxydopamine. Monoamine oxidase A and B inhibitors, tranylcypromine and clorgyline but not l-deprenyl increased the extent of 6-hydroxydopamine induced inhibition of complex I. Thus, 6-hydroxydopamine itself and not its oxidation products may be responsible for the neurotoxicity of this agent via inhibition of respiratory chain enzymes.

Dopamine neurotoxicity: inhibition of mitochondrial respiration.

Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen-derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose-dependent manner with LD50 = 90 micrograms. Norepinephrine was less effective with LD50 = 141 micrograms. The iron chelator desferrioxamine completely protected against dopamine-induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine-induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron-induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC50 = 8 microM, and that of norepinephrine was twice as much (IC50 = 15 microM). Dopamine-induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine-induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine-induced inhibition of NADH dehydrogenase activity in a dose-dependent manner.