A common signaling pathway leading to degranulation in mast cells and its regulation by CCR1-ligand.

BACKGROUND: Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. METHODS: Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. RESULTS: All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. CONCLUSIONS: Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.

AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

As a master regulator for metabolic and energy homeostasis, AMPK controls the activity of metabolic enzymes and transcription factors in response to cellular ATP status. AMPK has been thus recognized as a main target for the regulation of cellular energy metabolism. Here, we report that AMPK can be down-regulated by the cullin-RING ubiquitin E3 ligase 4A (CRL4A) with cereblon (CRBN). CRL4A interacted with AMPK holoenzymes and mediated AMPKα-specific polyubiquitination for its proteasomal degradation through non-K48 polyubiquitin linkages. In the ubiquitination system, CRBN was required for efficient polyubiquitination of AMPKα subunits. Consistently, polyubiquitination of AMPKα subunits was reduced by inhibitors of CRL4A-CRBN. Physiologic function of AMPK down-regulation by CRL4-CRBN was also confirmed using mouse bone marrow-derived mast cells (BMMCs). The inactivation of CRL4A-CRBN in BMMC increased AMPK stability and suppressed secretion of allergic mediators via AMPK activation followed by MAPK inhibition. In addition, CRBN knockout of BMMC also decreased allergic responses in mice. Our results suggest that the CRL4A-CRBN axis could be a target for the regulation of AMPK-dependent responses.-Kwon, E., Li, X., Deng, Y., Chang, H. W., Kim, D. Y. AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

Esculentoside H inhibits colon cancer cell migration and growth through suppression of MMP-9 gene expression via NF-kB signaling pathway.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-ß-d-glucopyranosyl-(1→4)-ß-d-xylopyranosyl)-28-ß-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

The orphan nuclear receptor NR4A1 promotes FcεRI-stimulated mast cell activation and anaphylaxis by counteracting the inhibitory LKB1/AMPK axis.

BACKGROUND: Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. OBJECTIVES: The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. METHODS: To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. RESULTS: Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. CONCLUSIONS: Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.

Inhibition of cereblon by fenofibrate ameliorates alcoholic liver disease by enhancing AMPK.

Alcohol consumption exacerbates alcoholic liver disease by attenuating the activity of AMP-activated protein kinase (AMPK). AMPK is activated by fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, and inhibited by direct interaction with cereblon (CRBN), a component of an E3 ubiquitin ligase complex. Based on these preliminary findings, we investigated that CRBN would be up-regulated in the liver by alcohol consumption and that CRBN deficiency would ameliorate hepatic steatosis and pro-inflammatory responses in alcohol-fed mice by increasing AMPK activity. Wild-type, CRBN and PPARα null mice were fed an alcohol-containing liquid diet and administered with fenofibrate. Gene expression profiles and metabolic changes were measured in the liver and blood of these mice. Expression of CRBN, cytochrome P450 2E1 (CYP2E1), lipogenic genes, pro-inflammatory cytokines, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were increased in the Lieber-DeCarli alcohol-challenged mice. Fenofibrate attenuated the induction of CRBN and reduced hepatic steatosis and pro-inflammatory markers in these mice. Ablation of the gene encoding CRBN produced the same effect as fenofibrate. The increase in CRBN gene expression by alcohol and the reduction of CRBN expression by fenofibrate were negated in PPARα null mice. Fenofibrate increased the recruitment of PPARα on CRBN gene promoter in WT mice but not in PPARα null mice. Silencing of AMPK prevented the beneficial effects of fenofibrate. These results demonstrate that activation of PPARα by fenofibrate alleviates alcohol-induced hepatic steatosis and inflammation by reducing the inhibition of AMPK by CRBN. CRBN is a potential therapeutic target for the alcoholic liver disease.

Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 µM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

DNA Topoisomerase Inhibitory Activity of Constituents from the Flowers of Inula japonica.

Fourteen compounds were isolated from the flowers of Inula japonica THUNB. (Asteraceae), including two new compounds, (1S,2S,4S,5S,8S,10R)-2-acetoxy-4,3-dihydroxy-pseudoguai-7(11)-en-12,8-olide (1) and (1S,2S,4S,5S,8S,10R)-2,4,13-trihydroxy-pseudoguai-7(11)-en-12,8-olide (2), and twelve known compounds, budlein B (3), 6ß-hydroxytomentosin (4), 6-deacetoxybritanin (5), 4-epipulchellin (6), britanin (7), tomentosin (8), (+)-dihydroquercetin (9), (-)-syringaresinol (10), quercetagetin 3,4'-dimethyl ether (11), luteolin (12), britanin G (13) and inuchinenolide C (14). Structures of 1 and 2 were determined based on one and two dimensional (1D)- and (2D)-NMR data and Mosher's esterification method. Compounds 9 and 12 showed inhibitory activities toward DNA topoisomerase I with IC50 values of 55.7 and 37.0 µM, respectively, compared to camptothecin (CPT) with an IC50 of 24.5 µM. Compounds 7-9 and 11-14 exhibited more potent inhibitory activity against topoisomerases II with IC50 values of 6.9, 3.8, 3.0, 6.9, 10.0, 14.7 and 13.8 µM, respectively, than that of etoposide (VP-16) with an IC50 of 26.9 µM. Compounds 4-7 and 10-14 exhibited weak cytotoxicities to the selected cancer cell lines.

Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells.

Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: ß1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis.

B-cell translocation gene 2 promotes hepatic hepcidin production via induction of Yin Yang 1.

Hepcidin is a peptide hormone secreted in the liver and plays a key role in maintaining iron homeostasis. Here, we demonstrate that B-cell translocation gene 2 (BTG2) is a key player in hepatic hepcidin regulation via induction of Yin Yang 1 (YY1). Hepatic hepcidin gene expression significantly enhanced by fasting states and glucagon exposure led to induction of gluconeogenic gene expression, and elevated serum hepcidin production in mice. Notably, overexpression of BTG2 using adenoviral system (Ad-BTG2) significantly elevated serum hepcidin levels via a significant induction of YY1 gene transcription. Immunoprecipitation studies demonstrated that BTG2 physically interacted with YY1 and recruited on the hepcidin gene promoter. Finally, ablation of hepatic BTG2 gene by gene silencing markedly attenuated the elevation of serum hepcidin production along with YY1 and hepcidin mRNA expression in fasting state. Likewise, forskolin (FSK)-stimulated hepcidin promoter activity was dramatically disrupted by endogenous BTG2 knockdown. Overall, our current study provides a novel molecular mechanism of BTG2-mediated induction of hepcidin gene expression, thereby contributing to a better understanding of the hepatic hepcidin production involved in iron homeostasis.

5-Lipoxygenase inhibitors suppress RANKL-induced osteoclast formation via NFATc1 expression.

5-Lipoxygenase synthesizes leukotrienes from arachidonic acid. We developed three novel 5-LO inhibitors having a benzoxazole scaffold as a potential anti-osteoclastogenics. They significantly suppressed RANKL-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, one compound, K7, inhibited the bone resorptive activity of osteoclasts. The anti-osteoclastogenic effect of K7 was mainly attributable to reduction in the expression of NFATc1, an essential transcription factor for osteoclast differentiation. K7 inhibited osteoclast formation via ERK and p38 MAPK, as well as NF-κB signaling pathways. K7 reduced lipopolysaccharide (LPS)-induced osteoclast formation in vivo, corroborating the in vitro data. Thus, K7 exerted an inhibitory effect on osteoclast formation in vitro and in vivo, properties that make it a potential candidate for the treatment of bone diseases associated with excessive bone resorption.

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus, Containing Lactose-Binding Molecules, on Human Leukemic K562 Cells.

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.

ERK1/2 antagonize AMPK-dependent regulation of FcεRI-mediated mast cell activation and anaphylaxis.

BACKGROUND: Extracellular signal-regulated kinases 1/2 (ERK1/2) make important contributions to allergic responses via their regulation of degranulation, eicosanoid production, and cytokine expression by mast cells, yet the mechanisms underlying their positive effects on FcεRI-dependent signaling are not fully understood. Recently, we reported that mast cell activation and anaphylaxis are negatively regulated by AMP-activated protein kinase (AMPK). However, little is known about the relationship between ERK1/2-mediated positive and the AMPK-mediated negative regulation of FcεRI signaling in mast cells. OBJECTIVE: We investigated possible interactions between ERK1/2 and AMPK in the modulation of mast cell signaling and anaphylaxis. METHODS: Wild-type or AMPKα2(-/-) mice, or bone marrow-derived mast cells obtained from these mice, were treated with either chemical agents or small interfering RNAs that modulated the activity or expression of ERK1/2 or AMPK to evaluate the functional interplay between ERK1/2 and AMPK in FcεRI-dependent signaling. RESULTS: The ERK1/2 pathway inhibitor U0126 and the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside similarly inhibited FcεRI-mediated mast cell signals in vitro and anaphylaxis in vivo. ERK1/2-specific small interfering RNA also mimicked this effect on FcεRI signals. Moreover, AMPKα2 knockdown or deficiency led to increased FcεRI-mediated mast cell activation and anaphylaxis that were insensitive to U0126 or activator 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside, suggesting that the suppression of FcεRI signals by the inhibition of the ERK1/2 pathway relies largely on AMPK activation. ERK1/2 controlled AMPK activity by regulating its subcellular translocation. CONCLUSIONS: ERK1/2 ablated the AMPK-dependent negative regulatory axis, thereby activating FcεRI signals in mast cells.

Progress in Studies on Rutaecarpine. II.--Synthesis and Structure-Biological Activity Relationships.

Rutaecarpine is a pentacyclic indolopyridoquinazolinone alkaloid found in Evodia rutaecarpa and other related herbs. It has a variety of intriguing biological properties, which continue to attract the academic and industrial interest. Studies on rutaecarpine have included isolation from new natural sources, development of new synthetic methods for its total synthesis, the discovery of new biological activities, metabolism, toxicology, and establishment of analytical methods for determining rutaecarpine content. The present review focuses on the synthesis, biological activities, and structure-activity relationships of rutaecarpine derivatives, with respect to their antiplatelet, vasodilatory, cytotoxic, and anticholinesterase activities.

Ganglioside GM3 participates in the TGF-ß1-induced epithelial-mesenchymal transition of human lens epithelial cells.

TGF-ß (transforming growth factor-ß)-induced EMT (epithelial-mesenchymal transition) induces the proliferation and migration of the HLE (human lens epithelial) cells. Ganglioside GM3, simple sialic-acid-containing glycosphingolipids on mammalian cell membranes, regulates various pathological phenomena such as insulin resistance and tumour progression. However, the relationship between ganglioside GM3 and TGF-ß-induced EMT in the HLE B-3 cells is poorly understood. In the present study we demonstrated that ganglioside GM3 was involved in TGF-ß1-induced EMT in HLE B-3 cells. Our results indicated that the expression of ganglioside GM3 and GM3 synthase mRNA were significantly increased in TGF-ß1-induced HLE B-3 cells. Reporter gene analysis also demonstrated that transcriptional activation of the GM3 synthase gene was regulated by Sp1 (specificity protein 1) in HLE B-3 cells upon TGF-ß1 stimulation. Interestingly, the inhibition of ganglioside GM3 expression by d-PDMP [d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol] and GM3 synthase shRNA (short hairpin RNA) resulted significantly in the suppression of cell migration and EMT-related signalling in HLE B-3 cells stimulated by TGF-ß. Furthermore, exogenous treatment of ganglioside GM3 rescued the expression of EMT molecules and cell migration suppressed by the depletion of ganglioside GM3 in TGF-ß1-induced HLE B-3 cells. We also found that ganglioside GM3 interacted with TGFßRs (TGF-ß receptors) in TGF-ß1-induced HLE B-3 cells. Taken together, these results suggest that ganglioside GM3 induced by TGF-ß1 regulates EMT by potential interaction with TGFßRs.

AMP-activated protein kinase negatively regulates FcεRI-mediated mast cell signaling and anaphylaxis in mice.

BACKGROUND: Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation, followed by inhibitory signals that turn off the activating signals. However, the overall view of negative signals in mast cells is still incomplete. Although AMP-activated protein kinase (AMPK), which is generally known as a regulator of energy metabolism, is also associated with anti-inflammation, little is known about the role of AMPK in mast cells. OBJECTIVES: We investigated the role of AMPK and its regulatory mechanism in mast cells. METHOD: The roles of AMPK in FcεRI-dependent activation of bone marrow-derived mast cells (BMMCs) were evaluated by using chemical agents, small interfering RNAs (siRNAs), or adenovirus that modulated the activity or expression of AMPK signaling components. In addition, AMPKα2(-/-) mice were used to verify the role of AMPK in anaphylactic models. RESULTS: FcεRI signaling and associated effector functions in BMMCs were suppressed by the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) and were conversely augmented by siRNA knockdown of AMPKα2 or liver kinase B1 (LKB1), an upstream kinase of AMPK. Furthermore, AMPKα2 deficiency led to increased FcεRI-mediated BMMC activation and anaphylaxis that were insensitive to AICAR, whereas enforced expression of AMPKα2 in AMPKα2(-/-) BMMCs reversed the hypersensitive FcεRI signaling to normal levels. Pharmacologic inhibition or siRNA knockdown of Fyn mimicked AMPK activation, suggesting that Fyn counterregulates the LKB1-AMPK axis. Mechanistically, Fyn controlled AMPK activity by regulating LKB1 localization. CONCLUSIONS: The Fyn-regulated LKB1-AMPK axis acts as a novel inhibitory module for mast cell activation, which points to AMPK activators as therapeutic drugs for allergic diseases.

Low molecular weight fucoidan improves endoplasmic reticulum stress-reduced insulin sensitivity through AMP-activated protein kinase activation in L6 myotubes and restores lipid homeostasis in a mouse model of type 2 diabetes.

Low molecular weight fucoidan (LMWF) is widely used to treat metabolic disorders, but its physiologic effects have not been well determined. In the present study, we investigated the metabolic effects of LMWF in obese diabetic mice (leptin receptor-deficient db/db mice) and the underlying molecular mechanisms involved in endoplasmic reticulum (ER) stress-responsive L6 myotubes. The effect of LMWF-mediated AMP-activated protein kinase (AMPK) activation on insulin resistance via regulation of the ER stress-dependent pathway was examined in vitro and in vivo. In db/db mice, LMWF markedly reduced serum glucose, triglyceride, cholesterol, and low-density lipoprotein levels, and gradually reduced body weights by reducing lipid parameters. Furthermore, it effectively ameliorated glucose homeostasis by elevating glucose tolerance. In addition, the phosphorylation levels of AMPK and Akt were markedly reduced by ER stressor, and subsequently, glucose uptake and fatty acid oxidation were also reduced. However, these adverse effects of ER stress were significantly ameliorated by LMWF. Finally, in L6 myotubes, LMWF markedly reduced the ER stress-induced upregulation of the mammalian target of rapamycin-p70S61 kinase network and subsequently improved the action of insulin via AMPK stimulation. Our findings suggest that AMPK activation by LMWF could prevent metabolic diseases by controlling the ER stress-dependent pathway and that this beneficial effect of LMWF provides a potential therapeutic strategy for ameliorating ER stress-mediated metabolic dysfunctions.

Pinusolide improves high glucose-induced insulin resistance via activation of AMP-activated protein kinase.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a crucial role in the maintenance of cellular energy homeostasis, and several natural compounds that activate AMPK possibly enhance glucose uptake by muscle cells. In this study, we found that pinusolide stimulated AMPK phosphorylation and glucose uptake and these effects were significantly reduced by siRNA LKB1 or compound C, suggesting that enhanced glucose uptake by pinusolide is predominantly accomplished via an LKB1-mediated AMPK activation pathway. An insulin resistance state was induced by exposing cells to 30mM glucose, as indicated by reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and glucose uptake. Under these conditions, the phosphorylation of AMPK and ACC were decreased. Surprisingly, disrupted insulin signaling and decreased AMPK activity by high glucose concentrations were prevented by pinusolide. Moreover, this treatment increased insulin-stimulated glucose uptake via AMPK activation. Taken together, our findings suggest a link between high glucose and insulin resistance in muscle cells, and provide further evidence that pinusolide attenuates blockade of insulin signaling by enhancing IRS-1 tyrosine phosphorylation by the activating the AMPK pathway. In addition, this study indicates the targeting of AMPK represents a new therapeutic strategy for hyperglycemia-induced insulin resistance and type 2 diabetes.

Tanshinone IIA improves endoplasmic reticulum stress-induced insulin resistance through AMP-activated protein kinase.

The aim of the present study was to determine the effect of Tanshinone IIA (Tan IIA) on endoplasmic reticulum (ER) stress-induced insulin resistance in L6 myotubes and db/db mice. ER stress markers, RNA-activated protein kinase-like ER resident kinase (PERK), JNK, and AMPK activity were determined in tunicamycin-treated L6 myotubes. Insulin resistance was monitored using glucose uptake assays in vitro and blood glucose levels in vivo. Tan IIA clearly suppressed the phosphorylations of PERK and JNK and potentiated insulin-mediated Akt phosphorylation as well as glucose uptake via AMPK activation under ER stress. Furthermore, these effects are completely abrogated by siRNA-mediated knockdown of AMPK or LKB1. In addition, Tan IIA reduced blood glucose levels and body weights in db/db mice without altering food intake. These findings suggest that Tan IIA enhances insulin sensitivity and improves glucose metabolic disorders by increasing AMPK activity and attenuating ER stress-induced insulin resistance.

Comparative proteome analysis of Tumor necrosis factor α-stimulated human Vascular Smooth Muscle Cells in response to melittin.

BACKGROUND: Bee venom has been used to relieve pain and to treat inflammatory diseases, including rheumatoid arthritis, in humans. To better understand the mechanisms of the anti-inflammatory and anti-atherosclerosis effect of bee venom, gel electrophoresis and mass spectrometry were used to identify proteins whose expression was altered in human Vascular Smooth Muscle Cells (hVSMCs) stimulated by tumor necrosis factor alpha after 12 h in the presence of melittin. RESULTS: To obtain valuable insights into the anti-inflammatory and anti-atherosclerosis mechanisms of melittin, two-dimensional (2-D) gel electrophoresis and MALDI-TOF/TOF were used. The proteome study, we showed 33 significant proteins that were differentially expressed in the cells treated with tumor necrosis factor alpha and melittin. Thirteen proteins were significantly increased in the cells treated with tumor necrosis factor alpha, and those proteins were reduced in the cells treated with melittin. Five of the proteins that showed increased expression in the cells treated with tumor necrosis factor alpha are involved in cell migration, including calreticulin, an essential factor of development that plays a role in transcription regulation. The proteins involved in cell migration were reduced in the melittin treated cells. The observed changes in the expression of GRP75, prohibitin, and a select group of other proteins were validated with reverse transcribed-PCR. It was confirmed that the observed change in the protein levels reflected a change in the genes level. In addition, the phosphorylation of EGFR and ERK was validated by analyzing the protein pathway. CONCLUSION: Taken together, these data established that the expression of some proteins was significantly changed by melittin treatment in tumor necrosis factor alpha stimulated the cells and provided insights into the mechanism of the melittin function for its potential use as an anti-inflammatory agent.

Manassantin B isolated from Saururus chinensis inhibits cyclooxygenase-2-dependent prostaglandin D2 generation by blocking Fyn-mediated nuclear factor-kappaB and mitogen activated protein kinase pathways in bone marrow derived-mast cells.

The authors investigated the effect of manassantin B (Man B) isolated from Saururus chinensis (S. chinensis) on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in mouse bone marrow derived-mast cells (BMMCs). Man B inhibited the generation of PGD2 dose-dependently by inhibiting COX-2 expression in immunoglobulin E (IgE)/Ag-stimulated BMMCs. To elucidate the mechanism responsible for the inhibition of COX-2 expression by Man B, the effects of Man B on the activation of nuclear factor-kappaB (NF-κB), a transcription factor essential and mitogen-activated protein kinases (MAPKs) for COX-2 induction, were examined. Man B attenuated the nuclear translocation of NF-κB p65 and its DNA-binding activity by inhibiting inhibitors of kappa Bα (IκBα) degradation and concomitantly suppressing IκB kinase (IKK) phosphorylation. In addition, Man B suppressed phosphorylation of MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38. It was also found that Man B suppressed Fyn kinase activation and consequent downstream signaling processes, including those involving Syk, Gab2, and Akt. Taken together, the present results suggest that Man B suppresses COX-2 dependent PGD2 generation by primarily inhibiting Fyn kinase in FcεRI-mediated mast cells.