Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress.

Abnormally high serum homocysteine levels have been associated with several disorders, including obesity, cardiovascular diseases or neurological diseases. Leptin is an anti-obesity protein and its action is mainly mediated by the activation of its Ob-R receptor in neuronal cells. The inability of leptin to induce activation of its specific signaling pathways, especially under endoplasmic reticulum stress, leads to the leptin resistance observed in obesity. The present study examined the effect of homocysteine on leptin signaling in SH-SY5Y neuroblastoma cells expressing the leptin receptor Ob-Rb. Phosphorylation of the signal transducer and activator of transcription (STAT3) and leptin-induced STAT3 transcriptional activity were significantly inhibited by homocysteine treatment. These effects may be specific to homocysteine and to the leptin pathway, as other homocysteine-related compounds, namely methionine and cysteine, have weak effect on leptin-induced inhibition of STAT3 phosphorylation, and homocysteine has no impact on IL-6-induced activation of STAT3. The direct effect of homocysteine on leptin-induced Ob-R activation, analyzed by Ob-R BRET biosensor to monitor Ob-R oligomerization and conformational change, suggested that homocysteine treatment does not affect early events of leptin-induced Ob-R activation. Instead, we found that, unlike methionine or cysteine, homocysteine increases the expression of the endoplasmic reticulum (ER) stress response gene, a homocysteine-sensitive ER resident protein. These results suggest that homocysteine may induce neuronal resistance to leptin by suppressing STAT3 phosphorylation downstream of the leptin receptor via ER stress.

Insulin Enhances Gene Expression of Midnolin, a Novel Genetic Risk Factor for Parkinson's Disease, via Extracellular Signal-Regulated Kinase, Phosphoinositide 3-Kinase and Multiple Transcription Factors in SH-SY5Y Cells.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Although many monogenic variants have been identified that cause familial PD, most cases are sporadic and the mechanisms of sporadic PD onset remain unclear. We previously identified midnolin (MIDN) as a novel genetic risk factor for PD in a Japanese population. MIDN copy number loss was strongly associated with sporadic PD, which was replicated in a British population. Furthermore, suppression of MIDN expression in rat pheochromocytoma cells inhibits neurite outgrowth and expression of Parkin ubiquitin ligase. However, the detailed molecular mechanisms of MIDN expression are unknown. We, therefore, investigated the molecular mechanism of MIDN expression in human neuroblastoma SH-SY5Y cells. We found that MIDN expression was promoted by insulin via extracellular-signal regulated kinase1/2 and phosphoinositide 3-kinase-dependent pathways. In addition, MIDN promoter activity was enhanced by mutations at transcription factor AP-2 consensus sequences and reduced by mutations at cAMP response element-binding protein and activator protein 1 (AP-1) consensus sequences. The dominant-negative cAMP response element-binding protein mutant did not block MIDN promoter activity, but both the pharmacological inhibitor and decoy oligodeoxynucleotide for AP-1 significantly blocked its activity. Additionally, DNA binding of c-FOS and c-JUN to the AP-1 consensus sequence in the MIDN promoter was enhanced by insulin as determined by chromatin immunoprecipitation, which suggested that AP-1 positively regulated MIDN expression. Taken together, this study reveals molecular mechanisms of MIDN gene expression induced by insulin in neuronal cells, and drugs which promote MIDN expression may have potential to be a novel medicine for PD. SIGNIFICANCE STATEMENT: We demonstrated that insulin promotes midnolin expression via extracellular-signal regulated kinase 1/2 and phosphoinositide 3-kinase pathways. Furthermore, we identified the important region of the MIDN promoter and showed that transcription factors, including activator protein 1, positively regulate MIDN expression, whereas transcription factor AP-2 negatively regulates basal and insulin-induced MIDN expression. We believe that our observations are important and that they contribute to the development of novel drugs to treat Parkinson's disease.

The possible role of biochanin A in ameliorating endoplasmic reticulum stress-induced leptin resistance.

Leptin plays an important role in energy intake and body weight homeostasis. Leptin is secreted mainly from white adipose tissue and circulates in the bloodstream, inhibiting food intake by activating the leptin receptor expressed on hypothalamic neurons. Recent studies have demonstrated leptin resistance as the main factor involved in the development of obesity. We and others have reported that leptin resistance is caused by endoplasmic reticulum (ER) stress due to the accumulation of unfolded protein in the ER. In the present study, we investigated whether isoflavones could affect ER stress and the subsequent development of leptin resistance. We showed that biochanin A, a family of isoflavones, strongly attenuated cell death induced by ER stress in neuronal cells, improved ER stress-induced impairments in leptin signaling, and suppressed ER stress-induced expression of glucose-regulated protein 78. These results suggest that biochanin A may have pharmacological properties that can ameliorate leptin resistance by reducing ER stress.

Anticancer effect of nor-wogonin (5, 7, 8-trihydroxyflavone) on human triple-negative breast cancer cells via downregulation of TAK1, NF-κB, and STAT3.

BACKGROUND: Nor-wogonin, a polyhydroxy flavone, has been shown to possess antitumor activity. However, the mechanisms responsible for its antitumor activity are poorly studied. Herein, we investigated the mechanisms of nor-wogonin actions in triple-negative breast cancer (TNBC) cells. METHODS: Effects of nor-wogonin on cell proliferation and viability of four TNBC cell lines (MDA-MB-231, BT-549, HCC70, and HCC1806) and two non-tumorigenic breast cell lines (MCF-10A and AG11132) were assessed by BrdU incorporation assays and trypan blue dye exclusion tests. Cell cycle and apoptosis analyses were carried out by flow cytometry. Protein expression was analyzed by immunoblotting. RESULTS: Nor-wogonin significantly inhibited the growth and decreased the viability of TNBC cells; however, it exhibited no or minimal effects in non-tumorigenic breast cells. Nor-wogonin (40 µM) was a more potent anti-proliferative and cytotoxic agent than wogonin (100 µM) and wogonoside (100 µM), which are structurally related to nor-wogonin. The antitumor effects of nor-wogonin can be attributed to cell cycle arrest via reduction of the expression of cyclin D1, cyclin B1, and CDK1. Furthermore, nor-wogonin induced mitochondrial apoptosis, (as evidenced by the increase in % of cells that are apoptotic), decreases in the mitochondrial membrane potential (ΔΨm), increases in Bax/Bcl-2 ratio, and caspase-3 cleavage. Moreover, nor-wogonin attenuated the expression of the nuclear factor kappa-B and activation of signal transducer and activator of transcription 3 pathways, which can be correlated with suppression of transforming growth factor-ß-activated kinase 1 in TNBC cells. CONCLUSION: These results showed that nor-wogonin might be a potential multi-target agent for TNBC treatment.

Possible involvement of 4-hydroxy-2-nonenal in the pathogenesis of leptin resistance in obesity.

Insensitivity to the antiobesity hormone, leptin, has been suggested to be involved in the pathogenesis of obesity. However, the pathological mechanisms underlying the development of leptin resistance are not well-understood. This study aimed to examine the pathological mechanisms of leptin resistance in obesity. In the present study, we found that 4-hydroxy-2-nonenal (4-HNE), an aldehyde, may be involved in the development of leptin resistance. The SH-SY5Y-Ob-Rb human neuroblastoma cell line, transfected to express the Ob-Rb leptin receptor stably, was treated with 4-HNE, and leptin-induced signal transduction was analyzed. We found that 4-HNE dose- and time-dependently inhibited leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, a major antiobesity signal of leptin. On the other hand, 4-HNE did not affect tyrosine phosphorylation of broad cellular proteins, suggesting that the inhibitory effect may be selective to leptin signaling. Mechanistically, 4-HNE induced the eukaryotic initiation factor 2α-CCAAT/enhancer-binding protein homologous protein arm of endoplasmic reticulum stress signaling, which may be involved in the pathogenesis of leptin resistance. Overall, these results suggest that 4-HNE may partly affect endoplasmic reticulum stress-induced unfolded protein response signaling and may be involved in the pathogenesis of leptin resistance.

Mechanisms of the action of adenine on anti-allergic effects in mast cells.

INTRODUCTION: Mast cells play an important role in allergic responses. METHODS: We herein demonstrated the mechanisms of inhibitory effect of adenine on IgE/antigen-induced degranulation and TNF-α release in mast cells. RESULTS: We found that these effects were dependent on the amino group of adenine because purine only weakly inhibited degranulation. Adenine also inhibited Ca2+ ionophore- and thapsigargin-induced degranulation, however, this inhibitory effect was weaker than that of the antigen. Therefore, the inhibitory effects of adenine on degranulation may be mediated before as well as after the Ca2+ raise under the antigen stimulus. Adenine inhibited antigen-induced Syk and the subsequent induction of AKT and ERK activation under FcϵRI-mediated signal. Adenine also attenuated antigen-induced increase in Ca2+ . Furthermore, adenine inhibited IgE/antigen-induced IKKα/ß activation, which is involved in degranulation. Finally, adenine protected mice against anaphylactic allergic responses in vivo. CONCLUSIONS: The present study revealed a key role of adenine in the attenuation of allergic responses through the inhibition of Syk-mediated signal transduction and IKK-mediated degranulation.

Loss of Stearoyl-CoA Desaturase-1 Activity Induced Leptin Resistance in Neuronal Cells.

The lack of response to leptin's actions in the brain, "leptin resistance," is one of the main causes of the pathogenesis of obesity. However, although high-fat diets affect sensitivity to leptin, the underlying mechanisms of leptin resistance are still an enigma. Here we examined the effect of excess saturated fatty acids (SFAs) on leptin signaling in human neuronal cells. Palmitate, the principle source of SFAs in diet, induced leptin resistance in a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb). We next investigated the function of stearoyl-CoA desaturase-1 (SCD1), an enzyme which converts SFAs into monounsaturated fatty acids (MUFAs), on leptin-induced signaling. We found that reduction of SCD1 activity, through SCD1 inhibition and knockdown, impairs leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human neuronal cells. Our findings suggested that SCD1 plays a key role in the pathophysiology of leptin resistance in neuronal cells associated with obesity.

Endospanin1 affects oppositely body weight regulation and glucose homeostasis by differentially regulating central leptin signaling.

The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.

Dehydroascorbic acid-induced endoplasmic reticulum stress and leptin resistance in neuronal cells.

Due to its anti-obesity effects, an adipocyte-derived hormone, leptin, has become important for the treatment of obesity. However, most obese subjects are in a state of leptin resistance, and endoplasmic reticulum (ER) stress is suggested to be involved in the pathophysiology of leptin resistance. Dehydroascorbic acid (DHAA), an oxidized form of vitamin C, was found to be increased in diabetes. In the present study, we investigated the possible effects of DHAA on the activation of ER stress and leptin resistance. A human neuroblastoma cell line, stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb), was treated with DHAA. We found that DHAA upregulated ER stress-related genes such as GRP78, CHOP, and spliced XBP1. Moreover, leptin-induced STAT3 phosphorylation was hindered by DHAA. These results suggested that increases in the levels of DHAA might be harmful to neurons, contributing to defective leptin-responsive signaling.

Inhibition of telomerase causes vulnerability to endoplasmic reticulum stress-induced neuronal cell death.

Endoplasmic reticulum (ER) stress is implicated in several diseases, such as cancer and neurodegenerative diseases. In the present study, we investigated the possible involvement of telomerase in ER stress-induced cell death. ER stress-induced cell death was ameliorated in telomerase reverse transcriptase (TERT) over-expressing MCF7 cells (MCF7-TERT cell). Telomerase specific inhibitor, BIBR1532, reversed the inhibitory effect of TERT on ER stress-induced cell death in MCF7-TERT cells. These findings suggest that BIBR1532 may specifically inhibit telomerase activity, thereby inducing cell death in ER stress-exposed cells. TERT was expressed in the SH-SY5Y neuroblastoma cell line. To analyze the possible involvement of telomerase in ER stress-induced neuronal cell death, we treated SH-SY5Y neuroblastoma cells with BIBR1532 and analyzed ER stress-induced cell death. We found that BIBR1532 significantly enhanced the ER stress-induced neuronal cell death. These findings suggest that inhibition of telomerase activity may enhance vulnerability to neuronal cell death caused by ER stress.

Key role of heat shock protein 90 in leptin-induced STAT3 activation and feeding regulation.

BACKGROUND AND PURPOSE: Leptin, an important regulator of the energy balance, acts on the brain to inhibit feeding. However, the mechanisms involved in leptin signalling have not yet been fully elucidated. Heat shock protein 90 (HSP90) is a molecular chaperone that is involved in regulating cellular homeostasis. In the present study, we investigated the possible involvement of HSP90 in leptin signal transduction. EXPERIMENTAL APPROACH: HEK293 and SH-SY5Y cell lines stably transfected with the Ob-Rb leptin receptor (HEK293 Ob-Rb, SH-SY5Y Ob-Rb) were used in the present study. Phosphorylation of JAK2 and STAT3 was analysed by western blotting. An HSP90 inhibitor was administered i.c.v. into rats and their food intake was analysed. KEY RESULTS: The knock-down of HSP90 in the HEK293 Ob-Rb cell line attenuated leptin-induced JAK2 and STAT3 signalling. Moreover, leptin-induced JAK2/STAT3 phosphorylation was markedly attenuated by the HSP90 inhibitors geldanamycin, radicicol and novobiocin. However, these effects were not mediated through previously known factors, which are known to be involved in the development of leptin resistance, such as suppressor of cytokine signalling 3 or endoplasmic reticulum stress. The infusion of an HSP90 inhibitor into the CNS blunted the anorexigenic actions of leptin in rats (male Wister rat). CONCLUSIONS AND IMPLICATIONS: HSP90 may be a novel factor involved in leptin-mediated signalling that is linked to anorexia.

Synthesis of a peptide that can translocate to the endoplasmic reticulum.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) leads to ER stress, which has been implicated in the development of diseases. In the present study, we synthesized a peptide that entered cells and translocated to the ER. This peptide possessed fluorescein isothiocyanate (FITC), HIV-TAT, mini-αA-crystallin, and KDEL sequences. We demonstrated that this peptide entered cells and translocated to the ER. Time course experiments revealed that this peptide existed in the ER of cos-7 cells for 16 h. Furthermore, we detected the full-length peptide in cells by fluorescent immunostaining followed by SDS-PAGE. The peptide also entered glial and neuronal cells. These results suggest that this peptide has the ability to enter cells and exert chaperone activity at the ER, and provide an insight into the development of new drugs.

Possible involvement of 15-deoxy-Δ(12,14) -prostaglandin J2 in the development of leptin resistance.

Obesity is a worldwide health problem that urgently needs to be solved. Leptin is an anti-obesity hormone that activates satiety signals to the brain. Evidence to suggest that leptin resistance is involved in the development of obesity is increasing; however, the molecular mechanisms involved remain unclear. We herein demonstrated that 15-deoxy-Δ(12,14) -prostaglandin J2 (15d-PGJ2 ) was involved in the development of leptin resistance. A treatment with 15d-PGJ2 inhibited the leptin-induced activation of signal transducer and activator of transcription 3 (STAT3) in neuronal cells (SH-SY5Y-Ob-Rb cells). Furthermore, the intracerebroventricular administration of 15d-PGJ2 reversed the inhibitory effects of leptin on food intake in rats. The peroxisome proliferator-activated receptor gamma (PPAR-γ) antagonist, GW9662, slightly reversed the inhibitory effects of 15d-PGJ2 on the leptin-induced activation of STAT3 in neuronal cells. The PPAR-γ agonist, rosiglitazone, also inhibited leptin-induced STAT3 phosphorylation. Therefore, the inhibitory effects of 15d-PGJ2 may be mediated through PPAR-γ. On the other hand, 15d-PGJ2 -induced leptin resistance may not be mediated by endoplasmic reticulum stress or suppressor of cytokine signaling 3. The results of the present study suggest that 15d-PGJ2 is a novel factor for the development of leptin resistance in obesity. Leptin resistance, an insensitivity to the actions of leptin, is involved in the development of obesity. Here, we found 15-deoxy-Δ(12,14) -prostaglandin J2 (15d-PGJ2 ) may be involved in the development of leptin resistance. The present results suggest that the 15d-PGJ2 may be a novel factor for the development of leptin resistance in obesity. 15d-PGJ2 , 15-Deoxy-Δ(12,14) -prostaglandin J2; STAT3, signal tranducer and activator of transcription 3.

Stress-induced neuroprotective effects of epiregulin and amphiregulin.

Members of the epidermal growth factor family play important roles in the regulation of cell growth, proliferation, and survival. However, the specific roles of each epidermal growth factor family member with respect to brain injury are not well understood. Gene chip assay screens have revealed drastic increases in the expression of the epidermal growth factor family members amphiregulin and epiregulin following lipopolysaccharide stimulation, which activates an immune response. Both immune activity and endoplasmic reticulum stress are activated during cerebral ischemia. We found that the expression levels of amphiregulin and epiregulin were significantly increased under conditions of cerebral ischemia. Because endoplasmic reticulum stress increased the expression of amphiregulin and epiregulin in glial cells, endoplasmic reticulum stress may be a key mediatory factor of pathophysiological activity. Recombinant epiregulin and amphiregulin proteins effectively inhibited endoplasmic reticulum stress and the subsequent induction of neuronal cell death. Therefore, the upregulation of the epidermal growth factor family members epiregulin and amphiregulin may play a critical role in preventing endoplasmic reticulum stress-induced cell death, thus providing a potential therapy for brain injury.

Leptin induced GRP78 expression through the PI3K-mTOR pathway in neuronal cells.

Leptin is a circulating hormone that plays a critical role in regulating energy expenditure and food intake. Evidence to suggest the involvement of endoplasmic reticulum (ER) stress in the development of obesity is increasing. To adapt against ER stress, cells trigger the unfolded protein response (UPR). The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that protects cells against ER stress by inducing protein folding. In the present study, we hypothesized that leptin may activate UPR and protect against ER stress associated with obesity. SH-SY5Y, a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb), was treated with leptin. We demonstrated that leptin induced GRP78 expression. We then validated the mechanism responsible for the leptin-induced expression of GRP78. Interestingly, leptin-induced GRP78 expression was not dependent on IRE1-XBP1 pathway. On the other hand, the PI3K inhibitor, LY294002, and mTOR inhibitor, rapamycin, inhibited the leptin-induced expression of GRP78. These results suggested that the leptin-induced expression of GRP78 may be dependent on the PI3K-mTOR pathway. Leptin specifically induced GRP78 because the induction of the ER-apoptotic marker, CHOP, was not detected in leptin-treated cells. Therefore, leptin may upregulate the expression of GRP78, thereby protecting against ER stress associated with obesity.

Therapeutic potential of flurbiprofen against obesity in mice.

Obesity is associated with several diseases including diabetes, nonalcoholic steatohepatitis (NASH), hypertension, cardiovascular disease, and cancer. Therefore, anti-obesity drugs have the potential to prevent these diseases. In the present study, we demonstrated that flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAID), exhibited therapeutic potency against obesity. Mice were fed a high-fat diet (HFD) for 6 months, followed by a normal-chow diet (NCD). The flurbiprofen treatment simultaneously administered. Although body weight was significantly decreased in flurbiprofen-treated mice, growth was not affected. Flurbiprofen also reduced the HFD-induced accumulation of visceral fat. Leptin resistance, which is characterized by insensitivity to the anti-obesity hormone leptin, is known to be involved in the development of obesity. We found that one of the possible mechanisms underlying the anti-obesity effects of flurbiprofen may have been mediated through the attenuation of leptin resistance, because the high circulating levels of leptin in HFD-fed mice were decreased in flurbiprofen-treated mice. Therefore, flurbiprofen may exhibit therapeutic potential against obesity by reducing leptin resistance.

TERT attenuated ER stress-induced cell death.

Tumor cells are frequently encountered in nutrient-deprived areas, though the mechanisms underlying their survival are unclear. In the present study, we found that depriving cells of glucose caused endoplasmic reticulum stress (ER stress) in a breast cancer cells line, MCF-7, and that specific activation of ER stress increased telomerase reverse transcriptase (TERT) expression. TERT expression would function in counteracting against the stress because over-expression of TERT diminished ER stress-induced cell death. Therefore, the results provide evidence for the underlying mechanisms of tumor progression in stressed conditions, highlighting that ER stress induces TERT expression to withstand environmental stress, a mechanism which we termed the "ER stress-TERT axis".

Caffeine attenuated ER stress-induced leptin resistance in neurons.

Exposing the endoplasmic reticulum (ER) to stress causes the accumulation of unfolded proteins, and subsequently results in ER stress. ER stress may be involved in various disorders such as obesity, diabetes, and neurodegenerative diseases. Leptin is an important circulating hormone, that inhibits food intake and accelerates energy consumption, which suppresses body weight gain. Recent studies demonstrated that leptin resistance is one of the main factors involved in the development of obesity. We and other groups recently reported the role of ER stress in the development of leptin resistance. Therefore, identifying drugs that target ER stress may be a promising fundamental strategy for the treatment of obesity. In the present study, we investigated whether caffeine could affect ER stress and the subsequent development of leptin resistance. We showed that caffeine exhibited chaperone activity, which attenuated protein aggregation. Caffeine also inhibited the ER stress-induced activation of IRE1 and PERK, which suggested the attenuation of ER stress. Moreover, caffeine markedly improved ER stress-induced impairments in the leptin-induced phosphorylation of STAT3. Therefore, these results suggest caffeine may have pharmacological properties that ameliorate leptin resistance by reducing ER stress.

ER stress upregulated PGE2/IFNγ-induced IL-6 expression and down-regulated iNOS expression in glial cells.

The disruption of endoplasmic reticulum (ER) function can lead to neurodegenerative disorders, in which inflammation has also been implicated. We investigated the possible correlation between ER stress and immune function using glial cells. We demonstrated that ER stress synergistically enhanced prostaglandin (PG) E2 + interferon (IFN) γ-induced interleukin (IL)-6 production. This effect was mediated through cAMP. Immune-activated glial cells produced inducible nitric oxide synthase (iNOS). Interestingly, ER stress inhibited PGE2 + IFNγ-induced iNOS expression. Similar results were obtained when cells were treated with dbcAMP + IFNγ. Thus, cAMP has a dual effect on immune reactions; cAMP up-regulated IL-6 expression, but down-regulated iNOS expression under ER stress. Therefore, our results suggest a link between ER stress and immune reactions in neurodegenerative diseases.

ER stress-mediated regulation of immune function under glucose-deprived condition in glial cells: up- and down-regulation of PGE2 + IFNγ-induced IL-6 and iNOS expressions.

Glucose metabolism plays central role in maintaining brain function. Under ischemic condition, where glucose levels were reduced, glial cells induce pro-inflammatory cytokine production. In the present study, we found prostaglandin (PG) E2+interferon (IFN) γ-induced interleukin (IL)-6 production was enhanced under glucose-deprived condition in the primary cultured glial cells. On the other hand, to our surprise, we found that PGE2+IFNγ-induced iNOS expression was attenuated under glucose-deprived condition. These dual effects would be mediated through endoplasmic reticulum (ER) stress, because we observed (1) up-regulation of GRP78 and CHOP under glucose-deprived condition, which was inhibited by chemical chaperon TMAO, and (2) treatment with TMAO inhibited IL-6 production under glucose-deprived condition. By activating theses responses glial cells may protect neurons because we observed increased neuronal cell viability in the immune-activated glial cell conditioned medium. Overall, our results suggest a link between ER stress and immune reactions under glucose-deprived condition in the glial cells.