Improved antioxidative defence protects insulin-producing cells against homocysteine toxicity.

Homocysteine (HC) is considered to play an important role in the development of metabolic syndrome complications. Insulin-producing cells are prone to HC toxicity and this has been linked to oxidative stress. However, the exact mechanisms remain unknown. Therefore it was the aim of this study to determine the nature of reactive oxygen species responsible for HC toxicity. Chronic exposure of RINm5F and INS1E insulin-producing cells to HC decreased cell viability and glucose-induced insulin secretion in a concentration-dependent manner and led to a significant induction of hydrogen peroxide generation in the cytosolic, but not the mitochondrial compartment of the cell. Cytosolic overexpression of catalase, a hydrogen peroxide detoxifying enzyme, provided a significant protection against viability loss and hydrogen peroxide generation, while mitochondrial overexpression of catalase did not protect against HC toxicity. Overexpression of CuZnSOD, a cytosolic superoxide dismutating enzyme, also protected against HC toxicity. However, the best protection was achieved in the case of a combined overexpression of CuZnSOD and catalase. Incubation of cells in combination with alloxan resulted in a significant increase of HC toxicity and an increase of hydrogen peroxide generation. Overexpression of CuZnSOD or catalase protected against the toxicity of HC plus alloxan, with a superior protection achieved again by combined overexpression. The results indicate that HC induces oxidative stress in insulin-producing cells by stimulation of superoxide radical and hydrogen peroxide generation in the cytoplasm. The low antioxidative defence status makes the insulin-producing cells very vulnerable to HC toxicity.

Ageing-induced changes in the redox status of peripheral motor nerves imply an effect on redox signalling rather than oxidative damage.

Ageing is associated with loss of skeletal muscle fibres, atrophy of the remaining fibres and weakness. These changes in muscle are accompanied by disruption of motor neurons and neuromuscular junctions although the direct relationship between the nerve and muscle degeneration is not understood. Oxidative changes have been implicated in the mechanisms leading to age-related loss of muscle mass and in degeneration of the central nervous system, but little is known about age-related changes in oxidation in specific peripheral nerves that supply muscles that are affected by ageing. We have therefore examined the sciatic nerve of old mice at an age when loss of tibialis anterior muscle mass and function is apparent. Sciatic nerve from old mice did not show a gross increase in oxidative damage, but electron paramagnetic resonance (EPR) studies indicated an increase in the activity of superoxide and/or peroxynitrite in the nerves of old mice at rest that was further exacerbated by electrical stimulation of the nerve to activate muscle contractions. Proteomic analyses indicated that specific redox-sensitive proteins are increased in content in the nerves of old mice that may reflect an adaptation to regulate the increased superoxide/peroxynitrite and maintain redox homoeostasis. Analysis of redox active cysteines showed some increase in reversible oxidation in specific proteins in nerves of old mice, but this was not universally seen across all redox-active cysteines. Detailed analysis of the redox-active cysteine in one protein in the nerve of old mice that is key to redox signalling (Peroxiredoxin 6, Cys 47) showed a minor increase in reversible oxidation that would be compatible with a change in its redox signalling function. In conclusion, the data presented indicate that sciatic nerve from old mice does not show a gross increase in oxidative damage similar to that seen in the TA and other muscles that it innervates. Our results indicate an adaptation to increased oxidation with minor changes in the oxidation of key cysteines that may contribute to defective redox signalling in the nerve.

Evaluation of the role of N-methyl-D-aspartate (NMDA) receptors in insulin secreting beta-cells.

The possibility that antagonism of N-methyl-D-aspartate (NMDA) receptors represent a novel drug target for diabetes prompted the current studies probing NMDA receptor function in the detrimental actions of homocysteine on pancreatic beta-cell function. Cellular insulin content and release, changes in membrane potential and intracellular Ca(2+) and gene expression were assessed following acute (20min) and long-term (18h) exposure of pancreatic clonal BRIN-BD11 beta-cells to known NMDA receptor modulators in the absence and presence of cytotoxic concentrations of homocysteine. As expected, acute or long-term exposure to homocysteine significantly suppressed basal and secretagogue-induced insulin release. In addition, NMDA reduced glucose-stimulated insulin secretion (GSIS). Interestingly, the selective NMDA receptor antagonist, MK-801, had no negative effects on GSIS. The effects of the NMDA receptor modulators were largely independent of effects on membrane depolarisation and increases of intracellular Ca(2+). However, combined culture of the NMDA antagonist, MK-801, with homocysteine did enhance intracellular Ca(2+) levels. Actions of NMDA agonists/antagonists and homocysteine on signal transduction pathways were independent of changes in cellular insulin content, cell viability, DNA damage or expression of key beta-cell genes. Taken together, the data support a role for NMDA receptors in controlling pancreatic beta-cell function. However, modulation of NMDA receptor function was unable to prevent the detrimental beta-cell effects of homocysteine.

Prolonged L-alanine exposure induces changes in metabolism, Ca(2+) handling and desensitization of insulin secretion in clonal pancreatic beta-cells.

Acute insulin-releasing actions of amino acids have been studied in detail, but comparatively little is known about the beta-cell effects of long-term exposure to amino acids. The present study examined the effects of prolonged exposure of beta-cells to the metabolizable amino acid L-alanine. Basal insulin release or cellular insulin content were not significantly altered by alanine culture, but acute alanine-induced insulin secretion was suppressed by 74% (P<0.001). Acute stimulation of insulin secretion with glucose, KCl or KIC (2-oxoisocaproic acid) following alanine culture was not affected. Acute alanine exposure evoked strong cellular depolarization after control culture, whereas AUC (area under the curve) analysis revealed significant (P<0.01) suppression of this action after culture with alanine. Compared with control cells, prior exposure to alanine also markedly decreased (P<0.01) the acute elevation of [Ca(2+)](i) (intracellular [Ca(2+)]) induced by acute alanine exposure. These diminished stimulatory responses were partially restored after 18 h of culture in the absence of alanine, indicating reversible amino-acid-induced desensitization. (13)C NMR spectra revealed that alanine culture increased glutamate labelling at position C4 (by 60%; P<0.01), as a result of an increase in the singlet peak, indicating increased flux through pyruvate dehydrogenase. Consistent with this, protein expression of the pyruvate dehydrogenase kinases PDK2 and PDK4 was significantly reduced. This was accompanied by a decrease in cellular ATP (P<0.05), consistent with diminished insulin-releasing actions of this amino acid. Collectively, these results illustrate the phenomenon of beta-cell desensitization by amino acids, indicating that prolonged exposure to alanine can induce reversible alterations to metabolic flux, Ca(2+) handling and insulin secretion.

Prolonged exposure to homocysteine results in diminished but reversible pancreatic beta-cell responsiveness to insulinotropic agents.

BACKGROUND: Plasma homocysteine levels may be elevated in poorly controlled diabetes with pre-existing vascular complications and/or nephropathy. Since homocysteine has detrimental effects on a wide diversity of cell types, the present study examined the effects of long-term homocysteine exposure on the secretory function of clonal BRIN-BD11 beta-cells. METHODS: Acute insulin secretory function, cellular insulin content and viability of BRIN-BD11 cells were assessed following long-term (18 h) exposure to homocysteine in culture. RT-PCR and Western blot analysis were used to determine the expression of key beta-cell genes and proteins. Cells were cultured for a further 18 h without homocysteine to determine any long-lasting effects. RESULTS: Homocysteine (250-1000 micromol/L) exposure reduced insulin secretion at both moderate (5.6 mmol/L) and stimulatory (16.7 mmol/L) glucose by 48-63%. Similarly, insulin secretory responsiveness to stimulatory concentrations of alanine, arginine, 2-ketoisocaproate, tolbutamide, KCl, elevated Ca2+, forskolin and PMA, GLP-1, GIP and CCK-8 were reduced by 11-62% following culture with 100-250 micromol/L homocysteine. These inhibitory effects could not simply be attributed to changes in cellular insulin content, cell viability, H2O2 generation or any obvious alterations of gene/protein expression for insulin, glucokinase, GLUT2, VDCC, or Kir6.2 and SUR1. Additional culture for 18 h in standard culture media after homocysteine exposure restored secretory responsiveness to all agents tested. CONCLUSION: These findings suggest that long-term exposure to high homocysteine levels causes a reversible impairment of pancreatic beta-cell insulinotropic pathways. The in vivo actions of hyperhomocysteinaemia on islet cell function merit investigation.