Chronic Ethanol Consumption-induced Pancreatic β-Cell Dysfunction and Apoptosis through Glucokinase Nitration and Its Down-regulation

Chronic ethanol consumption is known as an independent risk factor for type 2 diabetes, which is characterized by impaired glucose homeostasis and insulin resistance; however, there is a great deal of controversy concerning the relationships between alcohol consumption and the development of type 2 diabetes. We investigated the effects of chronic ethanol consumption on pancreatic β-cell dysfunction and whether generated peroxynitrite participates in the impaired glucose homeostasis. Here we show that chronic ethanol feeding decreases the ability of pancreatic β-cells to mediate insulin secretion and ATP production in coordination with the decrease of glucokinase, Glut2, and insulin expression. Specific blockade of ATF3 using siRNA or C-terminally deleted ATF3(ΔC) attenuated ethanol-induced pancreatic β-cell apoptosis or dysfunction and restored the down-regulation of glucokinase (GCK), insulin, and pancreatic duodenal homeobox-1 induced by ethanol. GCK inactivation and down-regulation were predominantly mediated by ethanol metabolism-generated peroxynitrite, which were suppressed by the peroxynitrite scavengers N^γ-monomethyl-l-arginine, uric acid, and deferoxamine but not by the S-nitrosylation inhibitor DTT, indicating that tyrosine nitration is the predominant modification associated with GCK down-regulation and inactivation rather than S-nitrosylation of cysteine. Tyrosine nitration of GCK prevented its association with pBad, and GCK translocation into the mitochondria results in subsequent proteasomal degradation of GCK following ubiquitination. This study identified a novel and efficient pathway by which chronic ethanol consumption may induce GCK down-regulation and inactivation by inducing tyrosine nitration of GCK, resulting in pancreatic β-cell apoptosis and dysfunction. Peroxynitrite-induced ATF3 may also serve as a potent upstream regulator of GCK down-regulation and β-cell apoptosis.     

SIRT3 Overexpression Attenuates Palmitate-Induced Pancreatic β-Cell Dysfunction

Abnormally high levels of circulating free fatty acids can lead to pancreatic islet β-cell dysfunction and apoptosis, contributing to β-cell failure in Type 2 diabetes. The NAD⁺-dependent protein deacetylase Sirtuin-3 (SIRT3) has been implicated in Type 2 diabetes. In this study, we tested whether SIRT3 overexpression affects palmitate-induced β-cell dysfunction in cells of line NIT1, which are derived from mouse pancreatic β-cells. Two different lengths of SIRT3 were overexpressed: full length SIRT3 (SIRT3LF), which was preferentially targeted to mitochondria and partially to the nucleus, and its N-terminal truncated form (SIRT3SF), which was located in the nucleus and cytoplasm. Overexpression of SIRT3LF and SIRT3SF using an adenoviral system alleviated palmitate-induced lipotoxicity such as reduction of cell viability and mitogen-activated protein kinase (MAPK) activation. Chronic exposure to low concentrations of palmitate suppressed glucose-stimulated insulin secretion, but the suppression was effectively reversed by overexpression of SIRT3LF or SIRT3SF. The mRNA levels of the endoplasmic reticulum (ER) stress responsive genes ATF4, GRP94 and FKBP11 were increased by palmitate treatment, but the increases were completely inhibited by SIRT3LF overexpression and less effectively inhibited by SIRT3SF overexpression. This result suggests that overexpression of SIRT3 inhibits induction of ER stress by palmitate. Collectively, we conclude that overexpression of SIRT3 alleviates palmitate-induced β-cell dysfunction.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.     

Resveratrol Inhibits Enterovirus 71 Replication and Pro-Inflammatory Cytokine Secretion in Rhabdosarcoma Cells through Blocking IKKs/NF-κB Signaling Pathway

Polydatin and resveratrol, as major active components in Polygonum cuspidatum, have anti-inflammatory, antioxidant and antitumor functions. However, the effect and mechanism of polydatin and resveratrol on enterovirus 71 (EV71) have not been reported. In this study, resveratrol revealed strong antiviral activity on EV71, while polydatin had weak effect. Neither polydatin nor resveratrol exhibited influence on viral attachment. Resveratrol could effectively inhibit the synthesis of EV71/VP1 and the phosphorylation of IKKα, IKKβ, IKKγ, IKBα, NF-κB p50 and NF-κB p65, respectively. Meanwhile, the remarkably increased secretion of IL-6 and TNF-α in EV71-infected rhabdosarcoma (RD) cells could be blocked by resveratrol. These results demonstrated that resveratrol inhibited EV71 replication and cytokine secretion in EV71-infected RD cells through blocking IKKs/NF-κB signaling pathway. Thus, resveratrol may have potent antiviral effect on EV71 infection.     

Dendrobium huoshanense Polysaccharides Prevent Inflammatory Response of Ulcerative Colitis Rat through Inhibiting the NF-κB Signaling Pathway

The polysaccharides of the Chinese herbal medicine Dendrobium huoshanense exhibit anti-inflammatory effects in multiple organs through regulating the immune responses. In the present study, we constructed ulcerative colitis (UC) model rats using dextran sulfate sodium to investigate the anti-inflammatory effects of D. huoshanense polysaccharides (DHP). After oral administration of DHP for two weeks, the indices of UC symptoms, including the ratio of colon weight to length, Disease Activity Index (DAI), and Colon Mucosal Damage Index (CMDI), all decreased significantly compared with the UC model group. The histological sections also revealed better cell orders in DHP treatments than in the UC model rats. Moreover, in treatment with high dose of DHP (200 mg/kg), the treatment efficacy arrived the similar levels to those in the treatment with 300 mg/kg sulfasalazine, which is a typical medicine to treat UC. These results indicated that DHP has a high efficacy to treat UC in model rats. Furthermore, serum levels of interleukin-1β, tumor necrosis factor-α, interleukin-17, and transforming growth factor-β were assessed using the enzyme linked immunosorbent assay (ELISA) method, and the levels of nuclear factor-κB in colon tissue sections were determined using the immunohistochemical method. The results showed that all these indices decreased significantly after administration of DHP in UC model rats, which might be the mechanisms underlying the DHP-suppressed UC inflammation. Overall, this study indicated that DHP might be directly used to treat UC and is a promising source to develop novel drugs against UC.     

Platycodin D Inhibits β-Amyloid-Induced Inflammation and Oxidative Stress in BV-2 Cells Via Suppressing TLR4/NF-κB Signaling Pathway and Activating Nrf2/HO-1 Signaling Pathway

Neurochemical Research - Alzheimer’s disease (AD) is a common neurodegenerative disease associated with deposition of β-amyloid peptide (Aβ). Platycodin D (PLD), a...     

Neurotropin Suppresses Inflammatory Cytokine Expression and Cell Death through Suppression of NF-κB and JNK in Hepatocytes

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.     

Full-length,Membrane-anchored TWEAK Can Function as a Juxtacrine Signaling Molecule and Activate the NF-κB Pathway

Tumor necrosis factor (TNF) family members are initially synthesized as type II transmembrane proteins, but some of these proteins are substrates for proteolytic enzymes that generate soluble cytokines with biological activity. TWEAK (TNF-like weak inducer of apoptosis), a member of the TNF family, is a multifunctional cytokine that acts via binding to a cell surface receptor named Fn14 (fibroblast growth factor-inducible 14). Studies conducted to date indicate that TWEAK-producing cells can co-express both membrane-anchored and soluble TWEAK isoforms, but there is little information on TWEAK proteolytic processing. Also, it is presently unclear whether membrane-anchored TWEAK, like soluble TWEAK, is biologically active. Here we show that full-length human TWEAK is processed intracellularly by the serine protease furin and identify TWEAK amino acid residues 90–93 as the predominant furin recognition site. In addition, we report that full-length, membrane-anchored TWEAK can bind the Fn14 receptor on neighboring cells and activate the NF-κB signaling pathway. Thus, TWEAK can act in a juxtacrine manner to initiate cellular responses, and this property may be important for TWEAK function during physiological wound repair and disease pathogenesis.     

The Overexpression of TGF-β and CCN2 in Intrauterine Adhesions Involves the NF-κB Signaling Pathway

Intrauterine adhesions (IUA) are a significant cause of menstrual disturbance and infertility, but their pathogenesis still remains unclear. Here, we investigated the expression of TGF-β and CCN2 in IUA endometrial tissue by immunohistochemistry, western blotting and qRT-PCR assays, and found the expression of TGF-β and CCN2 in the endometrial tissue of IUA was significantly increased compared to normal endometrium and uterine septum (P<0.01), suggesting that TGF-β and CCN2 may play an important role in the formation of IUA. Moreover, the activity of the NF-κB signaling pathway in endometrial tissue of IUA was also significantly enhanced compared to normal endometrial and uterine septum (P<0.01) and positively correlated with the expression of TGF-β and CCN2, which suggested that TGF-β and CCN2 expression may be involved in the NF-κB signaling pathway. Blocking the NF-κB signaling pathway using SN50 resulted in the reduced expression of TGF-β in RL95-2 cells, which confirmed the association of the NF-κB signaling pathway and TGF-β in endometrial cells. Additionally, the expression of TGF-β and CCN2 was associated with IUA recurrence, which provides a potential prognostic indictor for IUA. Together, these results demonstrated that TGF-β and CCN2 play an important role in IUA formation, whose mechanism was associated with the activation of the NF-κB signaling pathway.     

Qishenyiqi Protects Ligation-Induced Left Ventricular Remodeling by Attenuating Inflammation and Fibrosis via STAT3 and NF-κB Signaling Pathway

Aim

Qi-shen-yi-qi (QSYQ), a formula used for the routine treatment of heart failure (HF) in China, has been demonstrated to improve cardiac function through down-regulating the activation of the Renin-Angiotensin-Aldosterone System (RAAS). However, the mechanisms governing its therapeutic effects are largely unknown. The present study aims to demonstrate that QSYQ treatment can prevent left ventricular remodeling in heart failure by attenuating oxidative stress and inhabiting inflammation.

Methods

Sprague-Dawley (SD) rats were randomly divided into 6 groups: sham group, model group (LAD coronary artery ligation), QSYQ group with high dosage, middle dosage and low dosage (LAD ligation and treated with QSYQ), and captopril group (LAD ligation and treated with captopril as the positive drug). Indicators of fibrosis (Masson, MMPs, and collagens) and inflammation factors were detected 28 days after surgery.

Results

Results of hemodynamic alterations (dp/dt value) in the model group as well as other ventricular remodeling (VR) markers, such as MMP-2, MMP-9, collagen I and III elevated compared with sham group. VR was accompanied by activation of RAAS (angiotensin II and NADPHoxidase). Levels of pro-inflammatory cytokines (TNF-α, IL-6) in myocardial tissue were also up-regulated. Treatment of QSYQ improved cardiac remodeling through counter-acting the aforementioned events. The improvement of QSYQ was accompanied with a restoration of angiotensin II-NADPHoxidase-ROS-MMPs pathways. In addition, “therapeutic” QSYQ administration can reduce both TNF-α-NF-B and IL-6-STAT3 pathways, respectively, which further proves the beneficial effects of QSYQ.

Conclusions

Our study demonstrated that QSYQ protected LAD ligation-induced left VR via attenuating AngII -NADPH oxidase pathway and inhabiting inflammation. These findings provide evidence as to the cardiac protective efficacy of QSYQ to HF and explain the beneficial effects of QSYQ in the clinical application for HF.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Biomarkers of TGF-β Signaling Pathway and Prognosis of Pancreatic Cancer

Background

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

Method

We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

Results

The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

Conclusion

Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.     

MFHAS1 Is Associated with Sepsis and Stimulates TLR2/NF-κB Signaling Pathway Following Negative Regulation

Malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) has a potential immunoregulatory role dependent on Toll-like receptors (TLRs). TLR2, associated with deleterious systemic inflammation, cardiac dysfunction, and acute kidney injury, acts synergistically in sepsis. The role of MFHAS1 in targeting TLR2 involved in sepsis has not been examined thus far. This study aimed to examine the relationship of MFHAS1 and sepsis, and the effect of MFHAS1 on the TLR2 signaling pathway. Blood samples were collected from eight sepsis patients after surgery and eight patients undergoing selective surgery to determine blood MFHAS1 levels. HEK 293 cells, RAW 264.7 macrophages and THP-1 monocytes were used to confirm the effect of MFHAS1 on TLR2 signaling pathway. Our study showed that blood MFHAS1 was significantly elevated in septic patients, and MFHAS1 was more increased in mononuclear cells from septic patients. Pam3CSK4 (TLR2 ligand) was found to induce MFHAS1 production in RAW 264.7 murine macrophages and THP-1 human monocytes in a time-dependent manner. MFHAS1 has dual effects on TLR2 signaling pathway and inflammation, i.e., inhibitory effect at 6 hours, and then stimulatory effect after 24 hours through the activation of TLR2/NF-κB signaling pathway, and MFHAS1 induced the phosphorylation of JNK and p38 after TLR2 stimulation.     

OGDHL Is a Modifier of AKT-Dependent Signaling and NF-κB Function

Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multi-enzyme OGDH complex (OGDHC) whose malfunction is associated with neuro-degeneration. The essential role of this complex is in the degradation of glucose and glutamate and the OGDHL gene (one component of OGDHC) is down-regulated by promoter hypermethylation in many different cancer types. These properties suggest a potential growth modulating role of OGDHL in cancer; however, the molecular mechanism through which OGDHL exerts its growth modulating function has not been elucidated.Here, we report that restoration of OGDHL expression in cervical cancer cells lacking endogenous OGDHL expression suppressed cell proliferation, invasion and soft agar colony formation in vitro. Knockdown of OGDHL expression in cervical cancer cells expressing endogenous OGDHL had the opposite effect. Forced expression of OGDHL increased the production of reactive oxygen species (ROS) leading to apoptosis through caspase 3 mediated down-regulation of the AKT signaling cascade and decreased NF-κB phosphorylation. Conversely, silencing OGDHL stimulated the signaling pathway via increased AKT phosphorylation. Moreover, the addition of caspase 3 or ROS inhibitors in the presence of OGDHL increased AKT signaling and cervical cancer cell proliferation.Taken together, these data suggest that inactivation of OGDHL can contribute to cervical tumorigenesis via activation of the AKT signaling pathway and thus support it as an important anti-proliferative gene in cervical cancer.     

Oroxylin-A Rescues LPS-Induced Acute Lung Injury via Regulation of NF-κB Signaling Pathway in Rodents

Background and Purpose

Successful drug treatment for sepsis-related acute lung injury (ALI) remains a major clinical problem. This study was designed to assess the beneficial effects of post-treatment of oroxylin A (OroA), a flavonoid, in ameliorating lipopolysaccharides (LPS)-induced lung inflammation and fatality.

Experimental Approach

Rats were injected with LPS (10 mg/kg, iv) to induce ALI, and OroA was given (15 mg/kg, iv) 1 hr or 6 hrs after LPS challenge. Twenty four hrs after LPS challenge, biochemical changes in the blood and lung tissues, and morphological/histological alterations in the lung associated with inflammation and injury were examined. Therapeutic effect of OroA was assessed by measuring the survival rate in endotoxemic mice.

Key Results

LPS (10 mg/kg, iv) significantly altered WBC counts, elevated plasma tumor necrosis factor (TNF)-α and nitric oxide (NO), increased pulmonary edema, thickened alveolar septa, and decreased survival rate. These changes were ameliorated by OroA (15 mg/kg, iv) administered 1 hr or 6 hrs after LPS challenge. This post-treatment also significantly attenuated LPS-induced activation of nuclear factor-κB (NF-κB) and the release of high mobility group box 1 (HMGB1) in lung tissues. Furthermore, post-treatment with OroA (60 mg/kg, ip) administered 1 hr or 6 hrs after LPS challenge in mice significantly increased survival rate.

Conclusion and Implication

OroA administered after induction of ALI by LPS significantly prevent and revere lung tissues injuries with increased survival rate. Positive post-treatment effects of OroA suggest that OroA is a potentially useful candidate for managing lung inflammation in LPS-induced endotoxemia and septic shock.     

Pancreatic β-Cell Death,Regeneration and Insulin Secretion: Roles of Poly(ADP-Ribose) Polymerase and Cyclic ADP-Ribose

In the early 1980s, we proposed a unifying model for β-cell damage (The OKAMOTO model), in which poly(ADP-ribose) synthetase/ polymerase (PARP) activation plays an essential role in the consumption of NAD⁺, which leads to energy depletion and necrotic cell death. In 1984, we demonstrated that the administration of PARP inhibitors to 90% depancreatized rats induces islet regeneration. From the regenerating islet-derived cDNA library we isolated Reg (Regenerating Gene) and demonstrated that Reg protein induces βcell replication via the Reg receptor and ameliorates experimental diabetes. More recently, we showed that the combined addition of IL-6 and dexamethasone induces the Reg gene expression in β-cells and that PARP inhibitors enhance the expression. In 1993, we found that cyclic ADP-ribose (cADPR), a product synthesized from NAD⁺, is a second messenger for intracellular Ca⁺ mobilization for insulin secretion by glucose, and proposed a novel mechanism of insulin secretion, the CD38-cADPR signal system. Therefore, PARP inhibitors prevent β-cell necrosis, induce β-cell replication and maintain insulin secretion. In this paper, we would like to present a perspective view based on our studies concerning cell death, cell regeneration, and cell function, especially on insulin-producing pancreatic βcells, in the processes of which poly(ADPribose) synthetase/polymerase (PARP) and cyclic ADP-ribose (cADPR) are functioning.     

MST1 Negatively Regulates TNFα-Induced NF-κB Signaling through Modulating LUBAC Activity

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