The Combination of Oolonghomobisflavan B and Diallyl Disulfide Induces Apoptotic Cell Death via 67-kDa Laminin Receptor/Cyclic Guanosine Monophosphate in Acute Myeloid Leukemia Cells.

Diallyl disulfide (DADS) is a well-known principal functional component derived from garlic (Allium sativum) that has various health benefits. Previously, we identified a 67-kDa laminin receptor, a receptor for oolong tea polyphenol oolonghomobisflavan B (OHBFB). However, its molecular mechanisms still remain to be elucidated. Here, we show that DADS synergistically enhanced the effect of the oolong tea polyphenol oolonghomobisflavan B (OHBFB), which induces apoptosis in acute myeloid leukemia (AML) cancer cells without affecting normal human peripheral blood mononuclear cells (PBMCs). The underlying mechanism of OHBFB-induced anti-AML effects involves the upregulation of the 67-kDa laminin receptor/endothelial nitric oxide synthase/cyclic guanosine monophosphate (cGMP)/protein kinase c delta (PKCδ)/acid sphingomyelinase (ASM)/cleaved caspase-3 signaling pathway. In conclusion, we show that the combination of OHBFB and DADS synergistically induced apoptotic cell death in AML cells through activation of 67LR/cGMP/PKCδ/ASM signaling pathway. Moreover, in this mechanism, we demonstrate DADS may reduce the enzyme activity of phosphodiesterase, which is a negative regulator of cGMP that potentiates OHBFB-induced AML apoptotic cell death without affecting normal PBMCs.

Abietane Diterpenoids Isolated from Torreya nucifera Disrupt Replication of Influenza Virus by Blocking the Phosphatidylinositol-3-Kinase (PI3K)-Akt and ERK Signaling Pathway.

Although vaccines and antiviral drugs are available, influenza viruses continue to pose a significant threat to vulnerable populations globally. With the emergence of drug-resistant strains, there is a growing need for novel antiviral therapeutic approaches. We found that 18-hydroxyferruginol (1) and 18-oxoferruginol (2) isolated from Torreya nucifera exhibited strong anti-influenza activity, with 50% inhibitory concentration values of 13.6 and 18.3 µM against H1N1, 12.8 and 10.8 µM against H9N2, and 29.2 µM (only compound 2) against H3N2 in the post-treatment assay, respectively. During the viral replication stages, the two compounds demonstrated stronger inhibition of viral RNA and protein in the late stages (12-18 h) than in the early stages (3-6 h). Moreover, both compounds inhibited PI3K-Akt signaling, which participates in viral replication during the later stages of infection. The ERK signaling pathway is also related to viral replication and was substantially inhibited by the two compounds. In particular, the inhibition of PI3K-Akt signaling by these compounds inhibited viral replication by sabotaging influenza ribonucleoprotein nucleus-to-cytoplasm export. These data indicate that compounds 1 and 2 could potentially reduce viral RNA and viral protein levels by inhibiting the PI3K-Akt signaling pathway. Our results suggest that abietane diterpenoids isolated from T. nucifera may be potent antiviral candidates for new influenza therapies.

Phosphodiesterase 5 Inhibitor Potentiates Epigallocatechin 3-O-Gallate-Induced Apoptotic Cell Death via Activation of the cGMP Signaling Pathway in Caco-2 Cells.

Epigallocatechin 3-O-gallate (EGCG) is a predominant component in green tea with various health benefits. The 67 kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that is overexpressed in various types of cancer; 67LR was identified a cell surface EGCG target that plays a pivotal role in tumor growth, metastasis, and resistance to chemotherapy. However, the plasma concentration of EGCG is limited, and its molecular mechanisms remain unelucidated in colon cancer. In this study, we found that the phosphodiesterase 5 (PDE5) inhibitor, vardenafil (VDN), potentiates EGCG-induced apoptotic cell death in colon cancer cells. The combination of EGCG and VDN induced apoptosis via activation of the endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase Cδ signaling pathway. In conclusion, the PDE5 inhibitor, VDN, may reduce the intracellular PDE5 enzyme activity that potentiates EGCG-induced apoptotic cell death in Caco-2 cells. These results suggest that PDE5 inhibitors can be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death. Therefore, EGCG may be employed as a therapeutic candidate for colon cancer.

SMART SKY EYE System for Preliminary Structural Safety Assessment of Buildings Using Unmanned Aerial Vehicles.

The development of unmanned aerial vehicles (UAVs) is expected to become one of the most commercialized research areas in the world over the next decade. Globally, unmanned aircraft have been increasingly used for safety surveillance in the construction industry and civil engineering fields. This paper presents an aerial image-based approach using UAVs to inspect cracks and deformations in buildings. A state-of-the-art safety evaluation method termed SMART SKY EYE (Smart building safety assessment system using UAV) is introduced; this system utilizes an unmanned airplane equipped with a thermal camera and programmed with various surveying efficiency improvement methods, such as thermography, machine-learning algorithms, and 3D point cloud modeling. Using this method, crack maps, crack depths, and the deformations of structures can be obtained. Error rates are compared between the proposed and conventional methods.

Eriodictyol-Amplified 67-kDa Laminin Receptor Signaling Potentiates the Antiallergic Effect of O-Methylated Catechin.

(-)-Epigallocatechin-3-O-(3-O-methyl) gallate (1, EGCG3″Me), an antiallergic O-methylated catechin, is present in high quantities in the green tea cultivar "Benifuuki" (Camellia sinensis L.). Previous studies have shown that EGCG3″Me inhibited basophil degranulation mediated through the cell-surface 67-kDa laminin receptor (67LR), but the mechanisms are not fully elucidated. This study aimed to investigate the mechanisms underlying the inhibitory effect of EGCG3″Me on IgE/antigen (Ag)-mediated degranulation and the combined effect of EGCG3″Me with eriodictyol (2), a bioactive flavanone. EGCG3″Me inhibited ß-hexosaminidase release from the rat basophilic/mast cell line RBL-2H3 stimulated by IgE/Ag and induced acid sphingomyelinase (ASM) activity. This induction was inhibited by anti-67LR antibody treatment. The ASM-specific inhibitor desipramine inhibited EGCG3″Me-induced suppression of degranulation. The soluble guanylate cyclase (sGC) inhibitor NS2028 weakened the potency of EGCG3″Me, and the sGC activator BAY41-2272 suppressed degranulation. The ability of EGCG3″Me to induce ASM activity and inhibit degranulation was amplified by eriodictyol. Furthermore, oral administration of the lemon-peel-derived eriodyctiol-7-O-glucoside (3) potentiated the suppressive effect of EGCG3″Me-rich "Benifuuki" green tea on the IgE/Ag-induced passive cutaneous anaphylaxis (PCA) reaction in BALB/c mice. These results suggest that EGCG3″Me inhibits IgE/Ag-mediated degranulation by inducing the 67LR/sGC/ASM signaling pathway, and eriodictyol amplifies this signaling.

Src Mediates Epigallocatechin-3-O-Gallate-Elicited Acid Sphingomyelinase Activation.

Epigallocatechin-3-O-gallate (EGCG) is one of the major bioactive compounds known to be present in green tea. We previously reported that EGCG shows selective toxicity through activation of the protein kinase B (Akt)/cyclic guanosine monophosphate (cGMP)/acid sphingomyelinase (ASM) axis via targeting its receptor 67-kDa laminin receptor (67LR), which is overexpressed in cancer. However, little is known about upstream mechanisms of EGCG-elicited ASM activation. In this study we show that the proto-oncogene tyrosine-protein kinase Src, also known as c-src, plays a crucial role in the anticancer effect of EGCG. We showed that EGCG elicits phosphorylation of Src at Tyr 416, a crucial phosphorylation site for its activity, and that the pharmacological inhibition of Src impedes the upstream events in EGCG-induced cell death signaling including upregulation of Akt activity, increase in cGMP levels, and activation of ASM. Moreover, focal adhesion kinase (FAK), which is involved in the phosphorylation of Src, is colocalized with 67LR. EGCG treatment enhanced interaction of FAK and 67LR. Consistent with these findings, pharmacological inhibition of FAK significantly neutralized EGCG-induced upregulation of Akt activity and activation of ASM. Taken together, FAK/Src play crucial roles in the upstream signaling of EGCG.

Orexin A-induced inhibition of leptin expression and secretion in adipocytes reducing plasma leptin levels and hypothalamic leptin resistance.

Orexin A (OXA) is a neuropeptide associated with plasma insulin and leptin levels involved in body weight and appetite regulation. However, little is known about the effect of OXA on leptin secretion in adipocytes and its physiological roles. Leptin secretion and expression were analysed in 3T3-L1 adipocytes. Plasma leptin, adiponectin and insulin levels were measured by ELISA assay. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) levels in the hypothalamus were evaluated by western blotting. OXA dose-dependently suppressed leptin secretion from 3T3-L1 adipocytes by inhibiting its gene expression while facilitating adiponectin secretion. The leptin inhibition by OXA was mediated via orexin receptors (OXR1 and OXR2). In addition to the pathway via extracellular signal-regulated kinases, OXA triggered adenylyl cyclase-induced cAMP elevation, which results in protein kinase A-mediated activation of cAMP response element-binding proteins (CREB). Accordingly, CREB inhibition restored the OXA-induced downregulation of leptin gene expression and secretion. Exogenous OXA for 4 weeks decreased fasting plasma leptin levels and increased hypothalamic pSTAT3 levels in high-fat diet-fed mice, regardless of increase in body weight and food intake. These results suggest that high dose of OXA directly inhibits leptin mRNA expression and thus secretion in adipocytes, which may be a peripheral mechanism of OXA for its role in appetite drive during fasting. It may be also critical for lowering basal plasma leptin levels and thus maintaining postprandial hypothalamic leptin sensitivity.

Ablation of catalase promotes non-alcoholic fatty liver via oxidative stress and mitochondrial dysfunction in diet-induced obese mice.

Hydrogen peroxide (H2O2) produced endogenously can cause mitochondrial dysfunction and metabolic complications in various cell types by inducing oxidative stress. In the liver, oxidative and endoplasmic reticulum (ER) stress affects the development of non-alcoholic fatty liver disease (NAFLD). Although a link between both stresses and fatty liver diseases has been suggested, few studies have investigated the involvement of catalase in fatty liver pathogenesis. We examined whether catalase is associated with NAFLD, using catalase knockout (CKO) mice and the catalase-deficient human hepatoma cell line HepG2. Hepatic morphology analysis revealed that the fat accumulation was more prominent in high-fat diet (HFD) CKO mice compared to that in age-matched wild-type (WT) mice, and lipid peroxidation and H2O2 release were significantly elevated in CKO mice. Transmission electron micrographs indicated that the liver mitochondria from CKO mice tended to be more severely damaged than those in WT mice. Likewise, mitochondrial DNA copy number and cellular ATP concentrations were significantly lower in CKO mice. In fatty acid-treated HepG2 cells, knockdown of catalase accelerated cellular lipid accumulation and depressed mitochondrial biogenesis, which was recovered by co-treatment with N-acetyl cysteine or melatonin. This effect of antioxidant was also true in HFD-fed CKO mice, suppressing fatty liver development and improving hepatic mitochondrial function. Expression of ER stress marker proteins and hepatic fat deposition also increased in normal-diet, aged CKO mice compared to WT mice. These findings suggest that H2O2 production may be an important event triggering NAFLD and that catalase may be an attractive therapeutic target for preventing NAFLD.

Epigallocatechin-3-O-gallate induces acid sphingomyelinase activation through activation of phospholipase C.

Epigallocatechin-3-O-gallate (EGCG)-induced cyclic guanosine monophosphate (cGMP) plays a crucial role in EGCG-induced cell death in various types of cancer cells. However, little is known regarding the early molecular events after cGMP induction. In this study, we showed that cGMP induction is sufficient to induce the phosphorylation of protein kinase C delta (PKCδ) at Ser664, the crucial kinase for EGCG-induced activation of acid sphingomyelinase (ASM). Using a chemical inhibitor library, we revealed that the inhibitors of the negative regulators of diacylglycerol strongly increase the effect of EGCG. We also showed that EGCG treatment increased phospholipase C (PLC) activity, and the same results were obtained with cGMP inducer treatment. EGCG-induced ASM activation was completely suppressed by pharmacological inhibition of PLC. Collectively, EGCG-induced cGMP activated the cGMP/PLC/PKCδ/ASM signaling axis in multiple myeloma cells.

Green Tea Polyphenol EGCG Upregulates Tollip Expression by Suppressing Elf-1 Expression.

TLR signaling is critical to innate immune system regulation; however, aberrant TLR signaling is involved in several diseases, including insulin resistance, Alzheimer's disease, and tumor metastasis. Moreover, a recent study found that TLR-4 signaling pathway inhibition might be a target for the suppression of chronic inflammatory disorders. In this article, we show that the green tea polyphenol epigallocatechin-3-O-gallate (EGCG) increases the expression of Toll interacting protein, a strong inhibitor of TLR4 signaling, by suppressing the expression of E74-like ETS transcription factor 1 (Elf-1). A mechanistic study revealed that EGCG suppressed Elf-1 expression via protein phosphatase 2A/cyclic GMP (cGMP)-dependent mechanisms. We also confirmed that orally administered EGCG and a cGMP inducer upregulated Toll interacting protein expression, increased intracellular levels of cGMP in macrophages, and suppressed Elf-1 expression. These data support EGCG and a cGMP inducer as potential candidate suppressors of TLR4 signaling.

Meridianin C inhibits the growth of YD-10B human tongue cancer cells through macropinocytosis and the down-regulation of Dickkopf-related protein-3.

Meridianin C is a marine natural product known for its anti-cancer activity. At present, the anti-tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD-8, YD-10B, YD-38 and HSC-3. Among the cells tested, meridianin C most strongly reduced the growth of YD-10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD-10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC-dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD-10B cells treated with meridianin C, pointing out that meridianin C-induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf-related protein-3 (DKK-3), a known negative regulator of macropinocytosis. A role for DKK-3 in regulating macropinocytosis in the YD-10B cells was confirmed by siRNA knockdown of endogenous DKK-3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK-3 overexpression, which resulted in a considerable inhibition of the meridianin C-induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti-proliferative effects via macropinocytosis in the highly tumorigenic YD-10B cell line and the effects are mediated in part through down-regulation of DKK-3.

The FOXO3/PGC-1ß signaling axis is essential for cancer stem cell properties of pancreatic ductal adenocarcinoma.

In 95% of patients with pancreatic ductal adenocarcinoma, recurrence is observed following chemotherapy. Findings from several studies have indicated that cancer stem cells (CSCs) are resistant to anticancer agents and may be involved in cancer recurrence and metastasis. The CD44 protein is a major CSC marker, and CD44 also plays an indispensable role in the CSC properties in several cancers, including pancreatic cancer; however, no clinical approach exists to inhibit CD44 activity. Here, we have performed knock-in/knockdown experiments, and we demonstrate that the forkhead box O3 (FOXO3)/liver kinase B1 (LKB1)/AMP-activated protein kinase/peroxisome proliferator-activated receptor-γ co-activator-1ß (PGC-1ß)/pyruvate dehydrogenase-A1 pathway is essential for CD44 expression and CSC properties. We observed that patients exhibiting high pyruvate dehydrogenase-A1 expression have a poor prognosis. Systemic PGC-1ß knock-out mice are fertile and viable and do not exhibit an overt phenotype under normal conditions. This suggests that cGMP induction and PGC-1ß inhibition represent potential strategies for treating patients with pancreatic ductal adenocarcinoma.

Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation.

Tomatidine inhibits tumor necrosis factor-α-induced apoptosis in C2C12 myoblasts via ameliorating endoplasmic reticulum stress.

In this study, we examined the effect of tomatidine on tumor necrosis factor (TNF)-α-induced apoptosis in C2C12 myoblasts. TNF-α treatment increased cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) protein levels in a dose- and time-dependent manner. Pretreatment of cells with 10 µM tomatidine prevented TNF-α-induced apoptosis, caspase 3 cleavage, and PARP cleavage. Cells were treated with 100 ng/mL TNF-α for 24 h, and flow cytometry was utilized to assess apoptosis using annexin-V and 7-aminoactinomycin D. TNF-α up-regulated activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression. This effect was suppressed by pretreatment with tomatidine. Pretreatment with 4-phenylbutyric acid (a chemical chaperone) also inhibited TNF-α-induced cleavage of caspase 3 and PARP and up-regulation of ATF4 and CHOP expression. In addition, tomatidine-mediated inhibition of phosphorylation of c-Jun amino terminal kinase (JNK) attenuated TNF-α-induced cleavage of PARP and caspase 3. However, tomatidine did not affect NF-κB activation in TNF-α-treated C2C12 myoblast cells. Taken together, the present study demonstrates that tomatidine attenuates TNF-α-induced apoptosis through down-regulation of CHOP expression and inhibition of JNK activation.

γ-Tocotrienol upregulates aryl hydrocarbon receptor expression and enhances the anticancer effect of baicalein.

Previous studies have identified biomolecules that mediate the physiological actions of food factors, such as amino acids, vitamins, fatty acids, minerals, plant polyphenols, and lactobacilli, suggesting that our bodies are equipped with an innate system that senses which food factors are required to maintain our health. However, the effects of environmental factors on food factor sensing (FFS) remains largely unknown. Tocotorienols (T3s), which belongs to the vitamin E family, possess several physiological functions, including cholesterol lowering and neuroprotective effects. Here, we investigated the effects of naturally abundant γ-T3 on FFS-related gene expressions in melanoma using a DNA chip. Our results showed that γ-T3 increased the expression level of aryl hydrocarbon receptor (AhR), a sensing molecule to plant polyphenol baicalein. The co-treatment with γ-T3 and baicalein enhanced the anti-proliferative activity of baicalein, accompanied by the downstream events of AhR-activation induced by baicalein. These data suggest that γ-T3 upregulates AhR expression and enhances its sensitivity to baicalein.

Regulation of IGFBP-2 expression during fasting.

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα null mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between -511 bp and -499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα null mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

Orexin A regulates plasma insulin and leptin levels in a time-dependent manner following a glucose load in mice.

AIMS/HYPOTHESIS: Orexin A (OXA) is a neuropeptide implicated in the regulation of arousal status and energy metabolism. Orexin receptors are expressed not only in the central nervous system but also in the pancreas and adipose tissue. However, little is known about the physiological function of orexins. This study investigated the role of exogenous OXA in blood glucose control after glucose load in mice. In addition, the effect of OXA on insulin secretion was also identified in mouse pancreatic beta cells. METHODS: Insulin secretion and intracellular Ca(2+) levels were measured in perifused mouse islets. To investigate the effects of exogenous OXA on blood glucose levels in vivo, intraperitoneal glucose tolerance tests were performed after a subcutaneous injection of OXA in normal and high-fat diet-induced diabetic mice. RESULTS: OXA significantly potentiated glucose-stimulated insulin secretion in vitro, which increased intracellular Ca(2+) levels, mainly through adenylate cyclase and ryanodine receptor activation. This Ca(2+)-dependent insulinotropic effect of OXA was blocked in Epac2 (Rapgef4)-deficient beta cells. After a glucose load in mice, exogenous OXA decreased blood glucose levels, compared with the control, by enhancing plasma insulin and decreasing plasma glucagon levels. Additionally, OXA caused a delayed increase in plasma leptin levels, resulting in lower plasma insulin levels when blood glucose levels fell to baseline. CONCLUSIONS/INTERPRETATION: These results suggest that OXA might be a critical regulator of insulin, glucagon and leptin secretion in response to glucose. Thus, exogenous OXA might have therapeutic potential in improving blood glucose control in patients with type 2 diabetes.

Melatonin ameliorates ER stress-mediated hepatic steatosis through miR-23a in the liver.

The endoplasmic reticulum (ER) stress induces hepatic steatosis and inflammation in the liver. Although melatonin ameliorates ER stress-target genes, it remains unknown whether melatonin protects against hepatic steatosis as well as inflammation through regulation of miRNA. MicroRNAs have been identified as pivotal regulators in the field of gene regulation and their dysfunctions are a common feature in a variety of metabolic diseases. Especially, among miRNAs, miR-23a has been shown to regulate ER stress. Herein, we investigated the crucial roles of melatonin in hepatic steatosis and inflammation in vivo. Tunicamycin challenge caused increase of hepatic triglyceride and intracellular calcium levels through activation of ER stress, whereas these phenomena were partially disrupted by melatonin. We also demonstrated that expression of miR-23a stimulated with tunicamycin was rescued by melatonin treatment, resulting in reduced ER stress in primary hepatocytes. Overall, these results suggest a new function of melatonin that is involved in ameliorating ER stress-induced hepatic steatosis and inflammation by attenuating miR-23a. Melatonin may be useful as a pharmacological agent to protect against hepatic metabolic diseases due to its ability to regulate expression of miR-23a.

Melatonin prevents pancreatic ß-cell loss due to glucotoxicity: the relationship between oxidative stress and endoplasmic reticulum stress.

Prolonged hyperglycemia results in pancreatic ß-cell dysfunction and apoptosis, referred to as glucotoxicity. Although both oxidative and endoplasmic reticulum (ER) stresses have been implicated as major causative mechanisms of ß-cell glucotoxicity, the reciprocal importance between the two remains to be elucidated. The aim of this study was to evaluate the differential effect of oxidative stress and ER stress on ß-cell glucotoxicity, by employing melatonin which has free radical-scavenging and antioxidant properties. As expected, in ß-cells exposed to prolonged high glucose levels, cell viability and glucose-stimulated insulin secretion (GSIS) were significantly impaired. Melatonin treatment markedly attenuated cellular apoptosis by scavenging reactive oxygen species via its plasmalemmal receptor-independent increase in antioxidant enzyme activity. However, treatments with antioxidants alone were insufficient to recover the impaired GSIS. Interestingly, 4-phenylbutyric acid (4-PBA), a chemical chaperone that attenuate ER stress by stabilizing protein structure, alleviated the impaired GSIS, but not apoptosis, suggesting that glucotoxicity induces oxidative and ER stress independently. We found that cotreatment of glucotoxic ß-cells with melatonin and 4-PBA dramatically improved both their survival and insulin secretion. Taken together, these results suggest that ER stress may be the more critical mechanism for prolonged high-glucose-induced GSIS impairment, whereas oxidative stress appears to be more critical for the impaired ß-cell viability. Therefore, combinatorial therapy of melatonin with an ER stress modifier may help recover pancreatic ß-cells under glucotoxic conditions in type 2 diabetes.