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PubMed2000

Selenoorganic compound, ebselen, inhibits nitric oxide and tumor necrosis factor-alpha production by the modulation of jun-N-terminal kinase and the NF-kappab signaling pathway in rat Kupffer cells.

In response to the bacterial endotoxin, LPS, Kupffer cells are induced to express NO and TNF-alpha. These compounds are involved in hepatic inflammation/injury, especially that associated with endotoxic shock. In this study, we demonstrate that ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one), a selenoorganic compound, blocks LPS-induced NO and TNF-alpha production by cultured rat liver Kupffer cells. LPS can activate both the NF-kappaB signaling pathway and MAPK signal transduction pathways such as JNK and p38 MAPK. We find that ebselen inhibits LPS-induced NF-kappaB nuclear translocalization, and also suppresses the LPS-induced phosphorylation of JNK, but not the phosphorylation of p38 MAPK. This inhibition of signal transduction leads to a decrease in the transcription of TNF-alpha and the inducible isoform of NO. Furthermore, ebselen inhibits LPS-induced COX-2 expression, which is responsible for proinflammatory prostaglandin production, without affecting constitutive COX-1 expression. These data suggest the mechanism by which ebselen acts as an antiinflammatory agent, and also suggest that ebselen may be potent in preventing hepatic injury such as endotoxic shock, in which Kupffer cell activation has been implicated.

Animals↗
PubMed2006

Possible involvement of calcium signaling pathways in L-leucine-stimulated protein synthesis in L6 myotubes.

L-Leucine is known to stimulate protein synthesis in L6 myotubes. In the present study, we examined the possible involvement of calcium signaling pathways in the stimulation of protein synthesis induced by L-leucine in L6 myotubes. After 16 h of treatment with L-leucine-depleted medium, the re-addition of L-leucine for 4 h augmented protein synthesis by about 50% as compared with an L-leucine-depleted control. Ryanodine receptor antagonists almost completely abolished the stimulatory effect of L-leucine, while IP(3) receptor antagonists showed partial inhibition when added simultaneously with L-leucine. These results suggest the possibility that calcium signaling pathways are involved in L-leucine-stimulated protein synthesis.

Calcium Channels↗
PubMed2002

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation.

We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a rho0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNA(Lys) (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21(Waf1/Cip1), a p53-regulated cyclin-dependent kinase inhibitor.

Animals↗
PubMed2005

[Role of MyD88-dependent nuclear factor-kappaB signaling pathway in the development of cardiac hypertrophy in vivo].

OBJECTIVE: To investigate the role of MyD88-dependent nuclear factor-kappaB (NF-(B) activation signaling pathway in the development of cardiac hypertrophy in vivo. METHODS: Dominant negative myeloid differentiation protein (dn-MyD) 88 fragment was inserted into pShuttle plasmid and then fused into adenovirus so as to construct Ad5-dn-MyD88. Some Sprague-Dawley (SD) rats underwent banding of aorta (aorta binding group) and some SD rats underwent sham operation (sham operation group). Part of the rats in the aorta banding group were transfected with Ad5-dn-MyD88 into the myocardium tissue (Ad5-dn-MyD88 transfection group) or adenovirus expressing dnMyD88 (Ad5-GFP) (control group) so as to determine the effect of blocking down stream of MyD88 signaling on the development of cardiac hypertrophy. Three days after the hearts of some rats from the 4 groups were collected and Western blotting was used to detect the expression of dn-MyD88 protein and fluorescent microscopy was used to detect the expression of GFP. Three weeks after the beginning of experiment the hearts were collected to calculate the heart weight/body weight (HW/BW) ratio and extract the plasma protein and nuclear protein. Electrophoretic mobility shift assay (EMSA) was used to determine the NFkappaB binding activity. Western blotting was used to examine the phosphorylation of IkappaBalpha and IKKalpha/beta with appropriate specific anti-phospho antibodies. RESULTS: Flag and dn-MyD88 were effectively expressed 3 days after the transfection of Ad5-dn-MyD88 into the myocardium. Three weeks after the HW/BW ratio was 0.47 +/- 0.01 in the aorta banding group, significantly higher, by 37.8%, than that of the sham operation group (0.34 +/- 0.01, P < 0.01), and was 0.41 +/- 0.02 in the Ad5-dn-MyD88 transfection group, significantly lower, by 11.58%, than that of the aorta banding group (P < 0.01); the myocardial ANP protein expression level of the aorta binding group was significantly higher, by 43.5%, than that of the sham operation group (P < 0.01) and 36.2% higher than that of the Ad5-dnMyD88 transfection group (P < 0.01); the ANP/GAPDH in the aorta binding group was significantly higher than that of the sham operation group (P < 0.01) and that of the Ad5-dn-MyD88 transfection group (P < 0.01); the NF-kappaB binding activity in the myocardium of the aorta banding group was 9.94 +/- 1.58, significantly higher, by 144.8%, than that of the sham operation group (4.06 +/- 0.52, P < 0.01) and significantly lower, by 41.8%, than that of the Ad5-dn-MyD88 transfection group (5.79 +/- 0.52, P < 0.05); the phospho-(p-) IkappaBalpha level and p-IkappaBalpha/IkappaBalpha of the aorta binding group were significantly higher than those of the sham operation group (P < 0.01) and significantly higher, by 26.7%, than that of the Ad5-dn-MyD88 transfection group (P < 0.05); the p-IKKalphabeta/IKKalphabeta in the myocardium of the aorta binding group was significantly higher, by 318.0%, than that of the sham operation group (P < 0.01), and significantly higher, by 77.4%, than that of the Ad5-dn-MyD88 transfection group (P < 0.01). CONCLUSION: MyD88-dependent NFkappaB signaling is a novel pathway for inducing the development of cardiac hypertrophy in vivo and blocking MyD88 mediated signaling pathway attenuates the development of cardiac hypertrophy.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Cisplatin activates survival signals in UM-SCC-23 squamous cell carcinoma and these signal pathways are amplified in cisplatin-resistant squamous cell carcinoma.

cis-Diaminodichloroplatinum (II) (cisplatin) is one of the most effective anticancer drugs and is widely used for the treatment of squamous cell carcinoma (SCC). However, its efficacy is often limited due to the development of resistance. Although several factors implicated in cisplatin resistance have been identified, the resistance mechanisms in detail are not fully understood yet. In the present study, we have examined the implication of survival signaling pathways in cisplatin-resistance. Cisplatin induced activation of Ras and its downstream effector kinases, Raf/MEK/ERK in UM-SCC-23 human squamous cell carcinoma, suggesting that this anticancer drug activates survival signal pathway in addition to apoptosis signals. In cisplatin-resistant UM-SCC-23 in culture, which we have established, the protein levels of Ras, Raf-1 and MEK were drastically elevated compared to parent UM-SCC-23, and ERK and Akt signals were constitutively activated. U0126, an inhibitor for MEK and LY294002, an inhibitor for phosphatidylinositol 3-kinase (PI3K), sensitized resistant UM-SCC-23 to cisplatin-induced cell death. These results indicate that Raf/MEK/ERK and PI3K/Akt signal cascades may play a considerable role in cisplatin resistance in SCC.

Antineoplastic Agents↗
PubMed1998

Glucose activates both K(ATP) channel-dependent and K(ATP) channel-independent signaling pathways in human islets.

Insulin secretion by isolated islets of Langerhans from 19 human donors (9 women and 10 men) was studied in vitro to test the hypothesis that human islets contain both the K(ATP) channel-dependent and the K(ATP) channel-independent signaling pathways. The results demonstrated the presence of both of these major pathways of glucose signaling. Thus, insulin secretion was stimulated by high glucose concentrations, by the sulfonylurea tolbutamide, and by a depolarizing concentration of potassium chloride. Diazoxide, which activates the K(ATP) channel, completely blocked the stimulation of release by glucose. Stimulation of insulin release by tolbutamide, which inhibits the K(ATP) channel and depolarizes the beta-cell, and inhibition of glucose-stimulated release by diazoxide, which activates the channel and repolarizes the beta-cell, confirm the involvement of the K(ATP) channel-dependent pathway in glucose signaling. The participation of the K(ATP) channel-independent pathway in the stimulation of insulin release by glucose was demonstrated for the first time in human islets. This was done in two ways. The first method, in the presence of diazoxide, blocked the action of glucose on the K(ATP) channel in combination with a depolarizing concentration of KCl to raise [Ca2+]i. Under these conditions, glucose stimulated insulin release. A second method to demonstrate the involvement of the K(ATP) channel-independent pathway was to close the K(ATP) channels with tolbutamide. Again, with no possibility of further action on the K(ATP) channel, glucose stimulated insulin release. In a final series of experiments, glucose-stimulated insulin release was profoundly inhibited by somatostatin, clonidine, and prostaglandin E2, but not by galanin.

Adenosine Triphosphate↗
PubMed2006

Activation of the mTOR signalling pathway is required for pancreatic growth in protease-inhibitor-fed mice.

Cholecystokinin (CCK)-induced pancreatic growth in mice involves parallel increases in DNA and protein. The mammalian target of rapamycin (mTOR) signalling pathway regulates mRNA translation and its activation is implicated in growth of various tissues. The aim of this study was to elucidate whether mTOR activation is required for pancreatic growth in a mouse model of increased endogenous CCK release. In mice fed chow containing the synthetic protease inhibitor camostat, protein synthetic rates and phosphorylation of two downstream targets of mTOR, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the ribosomal protein S6 (S6), increased in comparison with fasted controls. The camostat-induced increases in protein synthesis and 4E-BP1 and S6 phosphorylation were almost totally abolished by administration of the mTOR inhibitor rapamycin 1 h prior to camostat feeding. In contrast, the phosphorylation of ERK1/2 and JNK and the expression of the early response genes c-jun, c-fos, ATF3 and egr-1 induced by camostat feeding were not affected by rapamycin. In mice fed camostat for 7 days, the ratio of pancreatic to body weight increased by 143%, but when rapamycin was administered daily this was reduced to a 22% increase. Changes in pancreatic mass were paralleled by protein and DNA content following camostat feeding and rapamycin administration. Moreover, while BrdU incorporation, an indicator of DNA synthesis, was increased to 448% of control values after 2 days of camostat feeding, rapamycin administration completely inhibited this increase. We conclude that the mTOR signalling pathway is required for CCK-induced cell division and pancreatic growth.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Nopp140 is a mediator of the protein kinase A signaling pathway that activates the acute phase response alpha1-acid glycoprotein gene.

The acute phase response (APR) in liver during inflammation is one of the well known examples for elucidating the signaling pathways that lead to the combinatorial regulation of gene expression. The APR is exemplified by alpha(1)-acid glycoprotein gene (agp) expression. A number of transcription factors, including CCAAT/enhancer-binding protein beta (C/EBPbeta), glucocorticoid receptor, cAMP-response element-binding protein (CREB), and Nopp140, are known to participate in its induction. The underlying mechanism of Nopp140 and other factors for regulating agp expression remains unclear. Here we demonstrate that protein kinase A (PKA)-dependent phosphorylation of Nopp140, together with C/EBPbeta, induces agp gene expression synergistically. The cooperative activation of the agp gene by Nopp140 and forskolin is sensitive to inhibition by PKI. Results from biochemical and functional characterizations of Nopp140 mutants defective in PKA phosphorylation sites suggest that PKA-dependent Nopp140 phosphorylation is important for its role in agp gene activation. Furthermore, maximal activation of the agp gene by PKA-phosphorylated Nopp140 depends on the presence of CREB and C/EBPbeta. The participation of CREB in the activation is, however, independent of its PKA-mediated phosphorylation. In summary, we demonstrate the existence of a novel Nopp140-mediated PKA signaling pathway that leads to the activation of agp, one of the major acute phase response genes.

Amino Acid Sequence↗
PubMed2005

The Notch signaling pathway is related to neurovascular progression of pancreatic cancer.

OBJECTIVE: To analyze the potential role of the Notch signaling pathway in pancreatic cancer angiogenesis and invasion. BACKGROUND: Angiogenesis, pain, and early neuroinvasion are clinical features of pancreatic cancer. Blood vessels and nerves develop together and use common routes through the organism. The Notch pathway (Notch-1/4, Jagged-1/2, Delta-1) appears crucial in this process. The current study analyzed the Notch pathway in pancreatic cancer and characterized its angiogenic and invasive effects. METHODS: Five PaCa cell lines were cultured for the in vitro experiments. Real-time quantitative RT-PCR was done to quantify mRNA expression in 31 human PaCa specimens, and immunohistochemistry was used to localize protein expression within tumor specimens. Activation of the Notch signaling was done by transfection of PaCa cells with a constitutive active Notch-1 mutant (Notch-IC). Overexpression of Jagged and Delta was achieved by transfection of full-length cDNA. Spheroid assays were used to study angiogenesis and ELISAs to measure VEGF, bFGF, and angiogenin expression. Matrigel invasion assays were used to analyze tumor cell invasion. RESULTS: Notch-3 and Notch-4 mRNA were significantly (P < 0.001) overexpressed in PaCa. Immunohistochemistry revealed protein accumulation of Notch-1 as well. All ligands were significantly up-regulated. A positive immunosignal of ligands was seen in nerves, blood vessels, and ductal tumor cells. Transfection of PaCa cells with the constitutive active Notch-IC mutant and with Jagged-1 revealed increased levels for VEGF. Concomitantly, recombinant Jagged-1 increased sprouting of endothelial cells in the spheroid assay. CONCLUSION: The Notch pathway most likely regulates neurovascular development in pancreatic cancer. Activation of this signaling pathway by constitutive Notch-1 mutants and by Jagged-1 causes an angiogenic and invasive tumor phenotype. Specific blockade of Notch signaling may therefore be beneficial for patients with pancreatic cancer.

Disease Progression↗
PubMed2003

Survival-signaling pathway as a promising target for cancer chemotherapy.

The serine/threonine kinase AKT, also known as PKB or RAC-PK, is a key molecule for protecting cells from undergoing apoptosis. Several studies have suggested that the AKT-mediated survival-signaling pathway is an attractive target for cancer chemotherapy: (1) the AKT pathway is relatively inactive in resting cells; (2) amplification of the AKT gene occurs in some tumors; (3) loss of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) is common in tumors and its loss constitutively activates AKT; (4) AKT is activated at the cancer invasion front. To clarify which drugs exhibit their cytotoxicity by inhibiting the AKT pathway, we screened anticancer drugs that could downregulate phospho-AKT levels and AKT kinase activity. We found that UCN-01 (7-hydroxystaurosporine), heat-shock protein 90 (HSP90) inhibitors, and topotecan (10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin) possessed the ability to interfere with the AKT pathway. UCN-01 directly suppressed upstream AKT kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) (IC(50) <33 nM) both in vitro and in tumor xenografts. HSP90 inhibitors and topotecan suppressed AKT activity via indirectly downregulating PDK1 and phosphatidylinositide-3-OH kinase activities. Transfection of the constitutively active AKT complementary DNA into cells attenuated the cytotoxic effects of the drugs, indicating that inhibition of the AKT pathway plays an important role in exerting their cytotoxic effects. These results strongly suggest that the AKT-mediated survival-signaling pathway is a promising and attractive target for cancer chemotherapy.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1999

Activation of integrin and ceramide signalling pathways can inhibit the mitogenic effect of insulin-like growth factor I (IGF-I) in human breast cancer cell lines.

Cell counting, cell cycle analysis and Western immunoblotting were used to examine the effects of non-apoptotic doses of a ceramide analogue, C2, and a synthetic arginine-glycine-aspartic acid (RGD)-containing peptide, RGD, in MCF-7 and T47D cells to determine whether activation of these signalling pathways could alter the mitogenic potential of insulin-like growth factor I (IGF-I). IGF-I alone increased total cell number in both cell lines, associated with a rise in the percentage of cells in the S-phase of the cell cycle and a co-incident increase in cyclin A production. Treatments alone had no effects on cell number or cyclin A production relative to controls. C2 inhibited IGF-I-induced mitogenesis in both lines, whereas RGD was only effective in the T47D line. Despite inhibition of cell proliferation, IGF-I stimulation of cells in S-phase and of cyclin A levels were unaffected; however, an IGF-I-induced increase in cyclin B1 levels was inhibited by 30%. Low-dose induction of integrin and ceramide signalling pathways causes cells to be blocked in S-phase, thereby inhibiting the normal cycle of events associated with the IGF-I-induced mitotic signal. Activating these pathways may not only restrict tumour growth by induction of apoptosis but they may also directly inhibit IGF-I-induced cell proliferation.

Breast Neoplasms↗
PubMed1998

Identification of a signaling pathway involved in calcium regulation of BDNF expression.

A signaling pathway by which calcium influx regulates the expression of the major activity-dependent transcript of BDNF in cortical neurons has been elucidated. Deletion and mutational analysis of the promoter upstream of exon III reveals that transactivation of the BDNF gene involves two elements 5' to the mRNA start site. The first element, located between 72 and 47 bp upstream of the mRNA start site, is a novel calcium response element and is required for calcium-dependent BDNF expression in both embryonic and postnatal cortical neurons. The second element, located between 40 and 30 bp upstream of the mRNA start site, matches the consensus sequence of a cAMP response element (CRE) and is required for transactivation of the promoter in postnatal but not embryonic neurons. The CRE-dependent component of the response appears to be mediated by CREB since it is part of the complex that binds to this CRE, and since dominant negative mutants of CREB attenuate transactivation of the promoter. A constitutively active mutant of CaM kinase IV, but not of CaM kinase II, leads to activation of the promoter in the absence of extracellular stimuli, and partially occludes calcium-dependent transactivation. The effects of CaM kinase IV on the promoter require an intact CRE. These mechanisms, which implicate CaM kinase IV and CREB in the control of BDNF expression, are likely to be centrally involved in activity-dependent plasticity during development.

Animals↗
PubMed1995

Adenosine A1 and A2b receptors coupled to distinct interactive signaling pathways in intestinal muscle cells.

Adenosine receptors and the signaling pathways to which they are coupled were examined in dispersed intestinal muscle cells. The receptors were characterized by their ability to induce contraction or relaxation, mobilize Ca2+ and stimulate or inhibit cAMP, in naive cells and in cells where only one receptor type was preserved by selective receptor protection. Adenosine elicited contraction and increased [Ca2+]i and cAMP; the contraction was mimicked by the A1 selective agonist, cyclopentyladenosine. A selective A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine, and pertussis toxin abolished contraction and the increase in [Ca2+]i and augmented the increase in cAMP. Conversely, a preferential A2 antagonist, 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-amine augmented contraction and the increase in [Ca2+]i and abolished the increase in cAMP; a cAMP-kinase inhibitor, Rp-cAMP[S], had a similar effect, augmenting contraction and the increase in [Ca2+]i. Adenosine elicited also relaxation of maximally contracted cells that increased or decreased in parallel with cAMP. The selective A2a agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine, was a very weak relaxant agent, and the selective A2a antagonist, 8-(3-chlorostyryl)caffeine, had no effect on adenosine-induced relaxation. In cells where only A1 receptors were preserved, the cAMP response to adenosine was abolished, although contraction and [Ca2+]i were increased to the same extent as when naive cells were treated with the A2 antagonist. Conversely, in cells where only A2 receptors were preserved, contraction and the increase in [Ca2+]i were abolished and the increase in cAMP was augmented to the same level as when naive cells were treated with the A1 antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1998

Differential kinetics for induction of interleukin-6 mRNA expression in murine peritoneal macrophages: evidence for calcium-dependent and independent-signalling pathways.

It is presently unclear what role elevations in intracellular calcium concentration ([Ca2+]i) play in the control of monokine secretion, or whether such alterations underlie the ability of physiologic stimuli to induce production of these important signalling molecules. To address these issues, we have performed experiments in murine peritoneal macrophages to determine whether lipopolysaccharide (LPS) or interferon gamma (IFN-gamma) initiate production of the proinflammatory monokine interleukin 6 (IL-6) concomitant with elevations in [Ca2+]i and with kinetics similar to that seen with known Ca2+ mobilizing agents. Alterations in [Ca2+]i after treatment with LPS, IFN-gamma, platelet activating factor (PAF), or thapsigargin were measured by fluorimetric methods. These effects were compared with the ability of each to induce IL-6 mRNA expression as measured by semiquantitative reverse-transcribed polymerase chain reactions. We report that neither LPS nor IFN-gamma elicited detectable elevations in [Ca2+]i but that both up-regulated expression of IL-6 mRNA expression within 60 min. In contrast, experiments using either thapsigargin or PAF showed rapid and dramatic elevations in [Ca2+]i with marked increases in IL-6 mRNA expression, as quickly as 15 min after initial exposure. Elevations in mRNA encoding IL-6 by thapsigargin and PAF were found to occur in a dose-dependent manner, mirroring their ability to elicit elevations in [Ca2+]i. These data demonstrate that LPS and IFN-gamma induce IL-6 message expression by means of Ca2+-independent signalling pathways. Furthermore, Ca2+-mobilizing agents that evoke monokine message expression do so far more rapidly than do LPS or IFN-gamma. Taken in concert, these data are consistent with the hypothesis that multiple signalling pathways exist by which production of proinflammatory monokines are initiated.

Animals↗
PubMed2000

Use of mass spectrometry to study signaling pathways.

Activation of cells by extracellular stimuli, such as growth factors, initiates a cascade of events involving posttranslational modifications, including phosphorylation, formation of protein complexes, and induction or repression of gene expression. Traditionally, genetic methods or specific biochemical assays have been used to identify molecules involved in signaling pathways. Lately, mass spectrometry, combined with elegant biochemical approaches, has become a powerful tool for identifying proteins and posttranslational modifications. With this protocol, we hope to bridge the gap between the biochemical and molecular aspects of signal transduction pathways and the mass spectrometric tools and techniques that are available to study them. We provide methods for large-scale cell culture and immunoprecipitation of tyrosine-phosphorylated proteins, silver staining of gels, trypsin digests, and protein identification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and nanoelectrospray tandem mass spectrometry. We discuss the special requirements for the identification of phosphorylation sites in proteins by mass spectrometry. We describe enrichment of phosphopeptides from unseparated peptide mixtures by immobilized metal affinity column (IMAC) and the use of phosphatases to identify phosphorylated peptides. We also discuss specialized methods, such as precursor ion scanning in the negative mode and direct sequencing of phosphopeptides in the positive mode. Our goal is to provide detailed methods in use today for proteomic applications in general and for receptor-mediated signaling pathways in particular.

Animals↗
PubMed2002

A mathematical model of metabolic insulin signaling pathways.

We develop a mathematical model that explicitly represents many of the known signaling components mediating translocation of the insulin-responsive glucose transporter GLUT4 to gain insight into the complexities of metabolic insulin signaling pathways. A novel mechanistic model of postreceptor events including phosphorylation of insulin receptor substrate-1, activation of phosphatidylinositol 3-kinase, and subsequent activation of downstream kinases Akt and protein kinase C-zeta is coupled with previously validated subsystem models of insulin receptor binding, receptor recycling, and GLUT4 translocation. A system of differential equations is defined by the structure of the model. Rate constants and model parameters are constrained by published experimental data. Model simulations of insulin dose-response experiments agree with published experimental data and also generate expected qualitative behaviors such as sequential signal amplification and increased sensitivity of downstream components. We examined the consequences of incorporating feedback pathways as well as representing pathological conditions, such as increased levels of protein tyrosine phosphatases, to illustrate the utility of our model for exploring molecular mechanisms. We conclude that mathematical modeling of signal transduction pathways is a useful approach for gaining insight into the complexities of metabolic insulin signaling.

Animals↗
PubMed2006

[Effects of pentoxifylline on hepatic nuclear factor-kappa B signaling pathway and insulin resistance in nonalcoholic steatohepatitis rats induced by fat-rich diet].

OBJECTIVE: To explore the effects of pentoxifylline (PTX) on nuclear factor-kappa B (NF-kB) signaling pathway, insulin receptor substrates (IRSs) and glucose transporter 2 (GLUT2) expressions in livers in a rat model of nonalcoholic steatohepatitis (NASH). METHODS: Rats fed a fat-rich diet for 4 weeks were randomly allocated into two groups; the model group rats (n = 12) were fed a high-fat diet alone and the PTX group rats (n = 12) were fed a high-fat diet plus PTX (100 mg x kg(-1)/d(-1)) in drinking water. Meanwhile, rats (n = 6) fed a standard diet from the start served as controls. All the rats were sacrificed at the end of the 24th week. Hepatic NF-kappaB binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of tumor necrosis factor (TNF) alpha and inhibitor kappaB (IkappaBalpha) proteins in livers were determined by Western blot. Messenger RNA of IRS-1, IRS-2 and GLUT2 expressions were examined by RT-PCR. RESULTS: NF-kappaB binding activity was higher in the model group than that in the controls, while it was lower in the PTX group compared with that in the model group. The expression of TNFalpha protein was markedly increased in the model group (vs. the control group) but decreased in the PTX group (vs. the model group). The expression of IkappaBaalpha protein was decreased in the model group (vs. the control group) but increased in the PTX group (vs. the model group) to a certain extent. IRS-2 mRNA expression was markedly increased in the model group, and significantly decreased in the PTX group when compared with the model group (P less than 0.01). CONCLUSIONS: PTX could influence NF-kappaB signaling pathway and IRS expression in livers of NASH rats, which might be involved in the improvement of hepatic insulin resistance.

Animals↗
PubMed2006

Signaling pathway of nitric oxide production induced by ginsenoside Rb1 in human aortic endothelial cells: a possible involvement of androgen receptor.

Ginsenosides have been shown to stimulate nitric oxide (NO) production in aortic endothelial cells. However, the signaling pathways involved have not been well studied in human aortic endothelial cells. The present study was designed to examine whether purified ginsenoside Rb1, a major active component of ginseng could actually induce NO production and to clarify the signaling pathway in human aortic endothelial cells. NO production was rapidly increased by Rb1. The rapid increase in NO production was abrogated by treatment with nitric oxide synthetase inhibitor, L-NAME. Rb1 stimulated rapid phosphorylation of Akt (Ser473), ERK1/2 (Thr202/Thr204) and eNOS (Ser1177). Rapid phosphorylation of eNOS (Ser1177) was prevented by SH-5, an Akt inhibitor or wortmannin, PI3-kinase inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. Interestingly, NO production and eNOS phosphorylation at Ser1177 by Rb1 were abolished by androgen receptor antagonist, nilutamide. The results suggest that PI3kinase/Akt and MEK/ERK pathways and androgen receptor are involved in the regulation of acute eNOS activation by Rb1 in human aortic endothelial cells.

Amino Acid Sequence↗