Reuterin isolated from the probiotic Lactobacillus reuteri promotes periodontal tissue regeneration by inhibiting Cx43-mediated the intercellular transmission of endoplasmic reticulum stress.

OBJECTIVE: The present study aimed to evaluate the effects of reuterin, a bioactive isolated from the probiotic Lactobacillus reuteri (L. reuteri) on periodontal tissue regeneration, and provide a new strategy for periodontitis treatment in the future. BACKGROUND: Data discussing the present state of the field: Probiotics are essential for maintaining oral microecological balance. Our previous study confirmed that probiotic L. reuteri extracts could rescue the function of mesenchymal stem cells (MSCs) and promote soft tissue wound healing by neutralizing inflammatory Porphyromonas gingivalis-LPS. Periodontitis is a chronic inflammatory disease caused by bacteria seriously leading to tooth loss. In this study, we isolated and purified reuterin from an extract of L. reuteri to characterize from the extracts of L. reuteri to characterize its role in promoting periodontal tissue regeneration and controlling inflammation in periodontitis. METHODS: Chromatographic analysis was used to isolate and purify reuterin from an extract of L. reuteri, and HNMR was used to characterize its structure. The inflammatory cytokine TNFα was used to simulate the inflammatory environment. Periodontal ligament stem cells (PDLSCs) were treated with TNFα and reuterin after which their effects were characterized using scratch wound cell migration assays to determine the concentration of reuterin, an experimental periodontitis model in rats was used to investigate the function of reuterin in periodontal regeneration and inflammation control in vivo. Real-time PCR, dye transfer experiments, image analysis, alkaline phosphatase activity, Alizarin red staining, cell proliferation, RNA-sequencing and Western Blot assays were used to detect the function of PDLSCs. RESULTS: In vivo, local injection of reuterin promoted periodontal tissue regeneration of experimental periodontitis in rats and reduced local inflammatory response. Moreover, we found that TNFα stimulation caused endoplasmic reticulum (ER) stress in PDLSCs, which resulted in decreased osteogenic differentiation. Treatment with reuterin inhibited the ER stress state of PDLSCs caused by the inflammatory environment and restored the osteogenic differentiation and cell proliferation functions of inflammatory PDLSCs. Mechanistically, we found that reuterin restored the functions of inflammatory PDLSCs by inhibiting the intercellular transmission of ER stress mediated by Cx43 in inflammatory PDLSCs and regulated osteogenic differentiation capacity. CONCLUSION: Our findings identified reuterin isolated from extracts of the probiotic L. reuteri, which improves tissue regeneration and controls inflammation, thus providing a new therapeutic method for treating periodontitis.

Reuterin in the healthy gut microbiome suppresses colorectal cancer growth through altering redox balance.

Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We find that microbial metabolites from healthy mice or humans are growth-repressive, and this response is attenuated in mice and patients with CRC. Microbial profiling reveals that Lactobacillus reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC. Reuterin alters redox balance, and reduces proliferation and survival in colon cancer cells. Reuterin induces selective protein oxidation and inhibits ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restricts colon tumor growth, increases tumor reactive oxygen species, and decreases protein translation in vivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.

Discovery of Novel Tacrine-Pyrimidone Hybrids as Potent Dual AChE/GSK-3 Inhibitors for the Treatment of Alzheimer's Disease.

Based on a multitarget strategy, a series of novel tacrine-pyrimidone hybrids were identified for the potential treatment of Alzheimer's disease (AD). Biological evaluation results demonstrated that these hybrids exhibited significant inhibitory activities toward acetylcholinesterase (AChE) and glycogen synthase kinase 3 (GSK-3). The optimal compound 27g possessed excellent dual AChE/GSK-3 inhibition both in terms of potency and equilibrium (AChE: IC50 = 51.1 nM; GSK-3ß: IC50 = 89.3 nM) and displayed significant amelioration on cognitive deficits in scopolamine-induced amnesia mice and efficient reduction against phosphorylation of tau protein on Ser-199 and Ser-396 sites in glyceraldehyde (GA)-stimulated differentiated SH-SY5Y cells. Furthermore, compound 27g exhibited eligible pharmacokinetic properties, good kinase selectivity, and moderate neuroprotection against GA-induced reduction in cell viability and neurite damage in SH-SY5Y-derived neurons. The multifunctional profiles of compound 27g suggest that it deserves further investigation as a promising lead for the prospective treatment of AD.

Proteomic Investigation of Glyceraldehyde-Derived Intracellular AGEs and Their Potential Influence on Pancreatic Ductal Cells.

Glyceraldehyde-derived advanced glycation end products (AGEs) play an important role in the pathogenesis of many diseases including cancer. Accumulation of intracellular AGEs could stimulate cancer induction and facilitate cancer progression. We evaluated the toxic effect of glyceraldehyde-derived intracellular AGEs on normal and malignant pancreatic ductal cells by assessing the cell viability, toxicity, and oxidative stress, followed by proteomic analysis. Our functional studies showed that pancreatic cancer cells (PANC-1 and MIA PaCa-2) were more resistant to glyceraldehyde treatment compared to normal pancreatic ductal epithelial cells (HPDE), while cytotoxicity effects were observed in all cell types. Furthermore, using 13C isotopic labeled glyceraldehyde, the proteomic data revealed a dose-dependent increment of the number of glycation adducts in both these cell types. HPDE cells showed a higher number of intracellular AGEs compared to cancer cells. At a molecular level, the glycations in the lysine residues of proteins showed a concurrent increase with the concentration of the glyceraldehyde treatment, while the arginine glycations appeared to be less affected by the glyceraldehyde doses. Further pathway analysis of these glycated proteins suggested that the glycated proteins participate in important biological processes that are major hallmarks of cancer initiation and progression, including metabolic processes, immune response, oxidative stress, apoptosis, and S100 protein binding.

The Effect of Glyceraldehyde-Derived Advanced Glycation End Products on ß-Tubulin-Inhibited Neurite Outgrowth in SH-SY5Y Human Neuroblastoma Cells.

Nutritional factors can affect the risk of developing neurological disorders and their rate of progression. In particular, abnormalities of carbohydrate metabolism in diabetes mellitus patients lead to an increased risk of neurological disorders such as Alzheimer's disease (AD). In this study, we investigated the relationship between nervous system disorder and the pathogenesis of AD by exposing SH-SY5Y neuroblastoma cells to glyceraldehyde (GA). We previously reported that GA-derived toxic advanced glycation end products (toxic AGEs, TAGE) induce AD-like alterations including intracellular tau phosphorylation. However, the role of TAGE and their target molecules in the pathogenesis of AD remains unclear. In this study, we investigated the target protein for TAGE by performing two-dimensional immunoblot analysis with anti-TAGE antibody and mass spectrometry and identified ß-tubulin as one of the targets. GA treatment induced TAGE-ß-tubulin formation and abnormal aggregation of ß-tubulin, and inhibited neurite outgrowth in SH-SY5Y cells. On the other hand, glucose-derived AGEs were also involved in developing AD. However, glucose did not make abnormal aggregation of ß-tubulin and did not inhibit neurite outgrowth. Understanding the underlying mechanism of TAGE-ß-tubulin formation by GA and its role in neurodegeneration may aid in the development of novel therapeutics and neuroprotection strategies.

Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation.

Antibiotic resistance is one of the world's greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.

Glyceraldehyde-Derived Pyridinium Evokes Renal Tubular Cell Damage via RAGE Interaction.

Glyceraldehyde-derived advanced glycation end products (glycer-AGEs) contribute to proximal tubulopathy in diabetes. However, what glycer-AGE structure could evoke tubular cell damage remains unknown. We first examined if deleterious effects of glycer-AGEs on reactive oxygen species (ROS) generation in proximal tubular cells were blocked by DNA-aptamer that could bind to glyceraldehyde-derived pyridinium (GLAP) (GLAP-aptamer), and then investigated whether and how GLAP caused proximal tubular cell injury. GLAP-aptamer and AGE-aptamer raised against glycer-AGEs were prepared using a systemic evolution of ligands by exponential enrichment. The binding affinity of GLAP-aptamer to glycer-AGEs was measured with a bio-layer interferometry. ROS generation was evaluated using fluorescent probes. Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GLAP-aptamer bound to glycer-AGEs with a dissociation constant of 7.7 × 10-5 M. GLAP-aptamer, glycer-AGE-aptamer, or antibodies directed against receptor for glycer-AGEs (RAGE) completely prevented glycer-AGE- or GLAP-induced increase in ROS generation, MCP-1, PAI-1, or RAGE gene expression in tubular cells. Our present results suggest that GLAP is one of the structurally distinct glycer-AGEs, which may mediate oxidative stress and inflammatory reactions in glycer-AGE-exposed tubular cells. Blockade of the interaction of GLAP-RAGE by GLAP-aptamer may be a therapeutic target for proximal tubulopathy in diabetic nephropathy.

Microbial Metabolite Signaling Is Required for Systemic Iron Homeostasis.

Iron is a central micronutrient needed by all living organisms. Competition for iron in the intestinal tract is essential for the maintenance of indigenous microbial populations and for host health. How symbiotic relationships between hosts and native microbes persist during times of iron limitation is unclear. Here, we demonstrate that indigenous bacteria possess an iron-dependent mechanism that inhibits host iron transport and storage. Using a high-throughput screen of microbial metabolites, we found that gut microbiota produce metabolites that suppress hypoxia-inducible factor 2α (HIF-2α) a master transcription factor of intestinal iron absorption and increase the iron-storage protein ferritin, resulting in decreased intestinal iron absorption by the host. We identified 1,3-diaminopropane (DAP) and reuterin as inhibitors of HIF-2α via inhibition of heterodimerization. DAP and reuterin effectively ameliorated systemic iron overload. This work provides evidence of intestine-microbiota metabolic crosstalk that is essential for systemic iron homeostasis.

Scleral Cross-Linking Using Glyceraldehyde for the Prevention of Axial Elongation in the Rabbit: Blocked Axial Elongation and Altered Scleral Microstructure.

BACKGROUND: This study aims to assess the efficacy of the scleral collagen cross-linking method using glyceraldehyde solution for prevention of lens-induced axial elongation in New Zealand rabbits and investigate the biochemical and microstructural changes that occur. METHODS: The right eyes of New Zealand rabbits aged seven weeks were randomly divided into three groups: the cross-linking group (n = 6), non-crosslinking group (n = 5), and untreated control group (n = 5). Eyes in cross-linking and non-crosslinking groups were treated with a -8.00 Diopter spherical lens over the course of two weeks. The cross-linking effects were achieved by a sub-Tenon's injection of 0.15 ml 0.5 M glyceraldehyde to eyes in the CL group. Ocular parameters were measured on the 1st, 7th, and 14th days. Biomechanical testing, light and electronic microscopy were used. RESULTS: Following the cross-linking treatment, eyes in the cross-linking group had a shorter axial length compared to those in the non-crosslinking group (p = 0.006). Collagen fibrils larger than 240 nm were observed in the scleral stroma of cross-linking group, which were absent in the scleral stroma of the non-crosslinking and untreated control group. The mean ultimate stress and Young's modulus was significantly greater in the cross-linking group compared to those in the non-crosslinking and untreated control group (p < 0.05). No histological damage observed in the retina or choroid. CONCLUSIONS: This study demonstrates that lens-induced axial elongation in rabbits can be effectively blocked by cross-linking using glyceraldehyde, with anatomical and mechanical modification and no deleterious effects.

Implications of alternative electron sinks in increased resistance of PSII and PSI photochemistry to high light stress in cold-acclimated Arabidopsis thaliana.

Exposure of control (non-hardened) Arabidopsis leaves to high light stress at 5 °C resulted in a decrease of both photosystem II (PSII) (45 %) and Photosystem I (PSI) (35 %) photochemical efficiencies compared to non-treated plants. In contrast, cold-acclimated (CA) leaves exhibited only 35 and 22 % decrease of PSII and PSI photochemistry, respectively, under the same conditions. This was accompanied by an accelerated rate of P700(+) re-reduction, indicating an up-regulation of PSI-dependent cyclic electron transport (CET). Interestingly, the expression of the NDH-H gene and the relative abundance of the Ndh-H polypeptide, representing the NDH-complex, decreased as a result of exposure to low temperatures. This indicates that the NDH-dependent CET pathway cannot be involved and the overall stimulation of CET in CA plants is due to up-regulation of the ferredoxin-plastoquinone reductase, antimycin A-sensitive CET pathway. The lower abundance of NDH complex also implies lower activity of the chlororespiratory pathway in CA plants, although the expression level and overall abundance of the other well-characterized component involved in chlororespiration, the plastid terminal oxidase (PTOX), was up-regulated at low temperatures. This suggests increased PTOX-mediated alternative electron flow to oxygen in plants exposed to low temperatures. Indeed, the estimated proportion of O(2)-dependent linear electron transport not utilized in carbon assimilation and not directed to photorespiration was twofold higher in CA Arabidopsis. The possible involvement of alternative electron transport pathways in inducing greater resistance of both PSII and PSI to high light stress in CA plants is discussed.

Unraveling the hydroxypropionaldehyde (HPA) system: an active antimicrobial agent against human pathogens.

The hydroxypropionaldehyde (HPA) system is a natural defense system synthesized by the probiotic bacterium Lactobacillus reuteri. To elucidate which of the molecules composing the HPA system (3-hydroxypropionaldehyde (3-HPA), reuterin (HPA dimer), and HPA hydrate) is responsible for the potent antimicrobial activity in biological systems, a combination of biochemical, genetic, and proteomic assays was used. The HPA system reacts with sulfhydryl-containing compounds such as cysteine and reduced glutathione (GSH) in solution. In situ, GSH knock-out Escherichia coli is significantly more susceptible to HPA-mediated cell death than E. coli wild type; GSH supplementation protects either bacteria from HPA attack. Proteomic analysis of HPA-treated bacteria ( Haemophilus influenzae ) revealed induction of redox- and heat shock-related proteins. A new antimicrobial mechanism of HPA is proposed, whereby the activity of HPA leads to depletion of free SH- groups in GSH and proteins through the action of 3-hydroxypropionaldehyde, causing an imbalance of the cellular redox status, ultimately resulting in cell death.

The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups.

Reuterin is an antimicrobial compound produced by Lactobacillus reuteri, and has been proposed to mediate, in part, the probiotic health benefits ascribed to this micro-organism. Despite 20 years of investigation, the mechanism of action by which reuterin exerts its antimicrobial effects has remained elusive. Here we provide evidence that reuterin induces oxidative stress in cells, most likely by modifying thiol groups in proteins and small molecules. Escherichia coli cells subjected to sublethal levels of reuterin expressed a set of genes that overlapped with the set of genes composing the OxyR regulon, which senses and responds to various forms of oxidative stress. E. coli cells mutated for oxyR were more sensitive to reuterin compared with wild-type cells, further supporting a role for reuterin in exerting oxidative stress. The addition of cysteine to E. coli or Clostridium difficile growth media prior to exposure to reuterin suppressed the antimicrobial effect of reuterin on these bacteria. Interestingly, interaction with E. coli stimulated reuterin production or secretion by L. reuteri, indicating that contact with other microbes in the gut increases reuterin output. Thus, reuterin inhibits bacterial growth by modifying thiol groups, which indicates that reuterin negatively affects a large number of cellular targets.

Gemfibrozil inhibits Legionella pneumophila and Mycobacterium tuberculosis enoyl coenzyme A reductases and blocks intracellular growth of these bacteria in macrophages.

We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of Legionella pneumophila and of 27 strains of wild-type and multidrug-resistant Mycobacterium tuberculosis in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited L. pneumophila fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxybutyrate. To assess the mechanism(s) of these effects, we cloned an L. pneumophila FabI enoyl reductase homolog that complemented for growth an Escherichia coli strain carrying a temperature-sensitive enoyl reductase and rendered the complemented E. coli strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this L. pneumophila FabI homolog, as well as M. tuberculosis InhA and E. coli FabI.

Crosslinking of scleral collagen in the rabbit using glyceraldehyde.

PURPOSE: To strengthen rabbit sclera in vivo using chemical crosslinking with glyceraldehyde for a scleral-based treatment of progressive myopia. SETTING: Department of Ophthalmology, Martin-Luther-University, Halle, Germany. METHODS: Five chinchilla rabbits were treated with sequential sub-Tenon injections of 0.15 mL 0.5 M glyceraldehyde into the superonasal quadrant of the right eye 5 times during 14 days. The rabbits were humanely killed and biomechanical stress-strain measurements of scleral strips from the treatment area were performed and compared with nontreated contralateral control sclera using a microcomputer-controlled biomaterial tester. The treated eyes were examined histologically by light microscopy to exclude possible adverse effects. RESULTS: Following the crosslinking treatment, the ultimate stress was 15.8 MPa +/- 6.0 (SD) versus 3.1 +/- 0.3 MPa in the controls (increase of 409.7%; P<.02), the Young modulus was 129.6 +/- 53.7 MPa versus 11.5 +/- 1.8 MPa in the controls (increase of 1027%, P<.01), and ultimate strain was 19.8% +/- 2.6% MPA versus 38.2% +/- 5.1% MPA in the controls (decrease of 48.2% P<.05). Histologically, mild side effects were found in the peripheral cornea adjacent to the treatment area, with some inflammatory infiltrate and moderate loss of keratocytes. CONCLUSIONS: Glyceraldehyde crosslinking of scleral collagen increased the scleral biomechanical rigidity efficiently. Glyceraldehyde can be easily applied by sequential parabulbar injections. There were no side effects on the retina, so the new method might become a treatment modality for strengthening scleral tissue to prevent progressive myopia.

Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria.

BACKGROUND: Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH) such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by Lb. reuteri SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed. RESULTS: Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator Escherichia coli (7.5-15 mM). Lactobacilli and Clostridium clostridioforme were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found. CONCLUSION: Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide.

Adenine nucleotide pattern in rat pancreatic islets exposed to nutrient secretagogues.

The effects of D-glucose, D-mannose, D-galactose, Dglyceraldehyde, pyruvate, L-lactate, 2-ketoisocaproate, L-leucine, and/or L-glutamine on the ATP and ADP content of rat isolated pancreatic islets were reevaluated in order to compare changes evoked by these nutrient secretagogues in the islet ATP content and ATP/ADP ratio to their effects upon insulin release. Although being compatible with the fuel concept for nutrient-stimulated insulin secretion, the results of this study also argue against the monolithic view that the adenine nucleotide pattern in islet cells represents the sole coupling factor between metabolic and more distal events in the process of nutrient-stimulated insulin release.

Advanced glycation end-products attenuate human mesenchymal stem cells and prevent cognate differentiation into adipose tissue, cartilage, and bone.

UNLABELLED: The impact of AGEs on human MSCs was studied. AGEs inhibited the proliferation of MSCs, induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders in aged and diabetic patients. INTRODUCTION: Advanced glycation end-products (AGEs) are accumulated on long-lived proteins of various tissues in advanced age and diabetes mellitus and have been implicated in chronic complication, including musculoskeletal disorders. Human mesenchymal stem cells (MSCs) potentially differentiate into mature musculoskeletal tissues during tissue repair, but the pathogenetic role of AGEs on MSCs is unclear. MATERIALS AND METHODS: AGEs were prepared by incubating BSA with glucose, glyceraldehydes, or glycolaldehyde (designated as AGE-1, AGE-2, or AGE-3, respectively). Proliferation, apoptosis, and reactive oxygen species (ROS) generation were assayed in AGE-treated cells. The expression of the receptor for AGE (RAGE) was examined by immunohistochemistry and Western blotting. Involvement of RAGE-mediated signaling was examined using a neutralizing antiserum against RAGE. Differentiation into adipose tissue, cartilage, and bone were morphologically and biochemically monitored with specific markers for each. RESULTS: AGE-2 and AGE-3, but not control nonglycated BSA and AGE-1, reduced the viable cell number and 5-bromo-2'deoxyuridine (BrdU) incorporation with increased intracellular ROS generation and the percentage of apoptotic cells. MSCs expressed RAGE and its induction was stimulated by AGE-2 and AGE-3. These AGEs inhibited adipogenic differentiation (assayed by oil red O staining, lipoprotein lipase production, and intracellular triglyceride content) and chondrogenic differentiation (assayed by safranin O staining and type II collagen production). On osteogenic differentiation, AGE-2 and AGE-3 increased alkaline phosphatase activity and intracellular calcium content; however, von Kossa staining revealed the loss of mineralization and mature bone nodule formation. The antiserum against RAGE partially prevented AGE-induced cellular events. CONCLUSION: AGE-2 and AGE-3 may lead to the in vivo loss of MSC mass and the delay of tissue repair by inhibiting the maturation of MSC-derived cells. The AGE-RAGE interaction may be involved in the deleterious effect of AGEs on MSCs.

D-Glyceraldehyde causes production of intracellular peroxide in pancreatic islets, oxidative stress, and defective beta cell function via non-mitochondrial pathways.

D-Glyceraldehyde (D-GLYC) is usually considered to be a stimulator of insulin secretion but theoretically can also form reactive oxygen species (ROS), which can inhibit beta cell function. We examined the time- and concentration-dependent effects of D-GLYC on insulin secretion, insulin content, and formation of ROS. We observed that a 2-h exposure to 0.05-2 mM D-GLYC potentiated glucose-stimulated insulin secretion (GSIS) in isolated Wistar rat islets but that higher concentrations inhibited GSIS. A 24-h exposure to 2 mm D-GLYC inhibited GSIS, decreased insulin content, and increased intracellular peroxide levels (2.14 +/- 0.31-fold increase, n = 4, p < 0.05). N-Acetylcysteine (10 mM) prevented the increase in intracellular peroxides and the adverse effects of d-GLYC on GSIS. In the presence of 11.1 but not 3.0 mm glucose, koningic acid (10 microM), a specific glyceraldehyde-3-phosphate dehydrogenase inhibitor, increased intracellular peroxide levels (1.88 +/- 0.30-fold increase, n = 9, p < 0.01) and inhibited GSIS (control GSIS = p < 0.001; koningic acid GSIS, not significant). To determine whether oxidative phosphorylation was the source of ROS formation, we cultured rat islets with mitochondrial inhibitors. Neither rotenone or myxothiazol prevented D-GLYC-induced increases in islet ROS. Adenoviral overexpression of manganese superoxide dismutase also failed to prevent the effect of D-GLYC to increase ROS levels. These observations indicate that exposure to excess D-GLYC increases reactive oxygen species in the islet via non-mitochondrial pathways and suggest the hypothesis that the oxidative stress associated with elevated D-GLYC levels could be a mechanism for glucose toxicity in beta cells exposed chronically to high glucose concentrations.

Islet secretory defect in insulin receptor substrate 1 null mice is linked with reduced calcium signaling and expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2b and -3.

Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary beta-cells. IRS-1 KO beta-cells exhibited a significantly shorter increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) than controls when briefly stimulated with glucose or glyceraldehyde and when l-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO beta-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca(2+) and O(2) consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca(2+) signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion.

Oxidative DNA damage by hyperglycemia-related aldehydes and its marked enhancement by hydrogen peroxide.

Increased risks of cancers and oxidative DNA damage have been observed in diabetic patients. Many endogenous aldehydes such as 3-deoxyglucosone and glyceraldehyde (GA) increase under hyperglycemic conditions. We showed that these aldehydes induced Cu(II)-mediated DNA damage, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation. GA had the strongest ability to damage DNA, and addition of low concentrations of H2O2 markedly enhanced the DNA damage. GA significantly increased 8-oxodG formation in human cultured cells (HL-60), and H2O2 enhanced it. We conclude that oxidative DNA damage by hyperglycemia-related aldehydes, especially GA, and marked enhancement of DNA damage by H2O2 may participate in diabetes-associated carcinogenesis.