Differential effects of ethanol on insulin-like growth factor-I receptor signaling

BACKGROUND: Activation of the insulin-like growth factor I receptor (IGF-IR) by its ligands IGF-I and IGF-II induces cell proliferation and protects against apoptosis. Ethanol inhibits IGF-IR tyrosine autophosphorylation, which subsequently interferes with the activation of key downstream signaling mediators including insulin-receptor substrate-1, phosphatidylinositol 3-kinase, and mitogen-activated protein (MAP) kinase. The ethanol-induced inhibition of IGF-IR signaling reduces mitogenesis and enhances apoptosis. In the current study, we demonstrate that the antiproliferative action of ethanol can be modulated by differential sensitivity of the autophosphorylation of the IGF-IR to ethanol. METHODS: A series of subclones was generated from 3T3 cells that express the human IGF-IR. RESULTS: There was considerable variability in the ability of ethanol to inhibit IGF-I-dependent IGF-IR tyrosine autophosphorylation and MAP kinase activation, despite equivalent IGF-IR expression. The IGF-IR was completely resistant to a high concentration of ethanol (150 mM) in several subclones. The sensitivity of IGF-IR autophosphorylation to ethanol correlated directly with the inhibition of IGF-I-mediated MAP kinase activation and cell proliferation. Resistant subclones exhibited features of the transformed phenotype including high MAP kinase activity, partial loss of contact inhibition, and the development of foci at confluency. The IGF-IR isolated from ethanol-resistant cells was similarly resistant to ethanol in autophosphorylation reactions in vitro, whereas ethanol inhibited the autophosphorylation of IGF-IR obtained from sensitive cells. CONCLUSIONS: Our findings are the first to demonstrate the modulation of ethanol sensitivity of a tyrosine kinase receptor, and they provide a molecular basis for differential effects of ethanol on cell proliferation.     

Ethanol-Induced Inhibition of Cell Proliferation Is Modulated by Insulin-Like Growth Factor-I Receptor Levels

Ethanol inhibits the tyrosine autophosphorylation of the insulin-like growth factor (IGF)-1 receptor, an action that correlates with the inhibition of IGF-I-stimulated cell proliferation [J. Biol. Chem . 268:21777–21782 (1993)l. In the current study, the IGF-I-dependent proliferation of mouse BALB/c3T3 cells was completely inhibited by ethanol, but the growth of BALB/c3T3 cells that overexpress the IGF-l receptor (p6 cells) was only partially inhibited by ethanol. BALB/ c3T3 cells that simultaneously overexpress both the IGF-I receptor and IGF-I were insensitive to growth inhibition by ethanol. In p6 cells, increasing concentrations of IGF-l overcame the inhibition of IGF-l receptor tyrosine autophosphorylation in the presence of ethanol. The importance of the IGF-I receptor as a specific target for ethanol was further investigated in C6 rat glioblastoma cells that respond mitogenically to both epidermal growth factor (EGF) and IGF-I. The mitogenic response of C6 cells to EGF was abrogated In cells expressing antisense mRNA to the IGF-l receptor. Thus, EGF action in these cells is dependent on activation of an IGF-I/lGF-I receptor au-tocrine pathway. Indeed, EGF stimulated an increase in IGF-l receptor levels by more than 100%. Ethanol completely inhibited the prollferation of C6 cells in response to either EGF or IGF-I. However, ethanol did not directly interfere with EGF receptor function, because EGF-induced cell proliferation was unaffected by ethanol when added exclusively during a 1-hr exposure to EGF. Ethanol did not interfere with the EGF-induced increase in IGF-I receptor expression. The addition of both EGF and IGF-I overcame the inhibitory action of ethanol. In conclusion, the potency of ethanol as an inhibitor of IGF-I-mediated cell proliferation correlates with the level of IGF-I receptors. In contrast to its effect on the IGF-I receptor, ethanol has no direct effect on EGF receptor activation.     

Insulin-like growth factor-I is an important antiapoptotic factor for rat leydig cells during postnatal development

The present investigation examines the influence of IGF-I and the role of IGF-I receptor (IGF-IR) in the apoptosis/survival of Leydig cells. Immunohistochemical analysis of the rat testis at different ages revealed that the level of the phosphorylated IGF-IR increases from birth to d 20 of postnatal life, remaining high in the adult testis. Western blotting revealed that this level is higher in Leydig cells isolated from 40-d-old than from 10- or 60-d-old rats. Application of the terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay revealed that IGF-I decreases the level of apoptosis in Leydig cells at all stages of development, and the selective inhibitor of IGF-IR, picropodophyllin, blocks this antiapoptotic effect. The mechanism underlying the antiapoptotic action of IGF-I involves the phosphatidylinositol 3-kinase/Akt pathway, and in immature Leydig cells, this growth factor enhances the expression of Bcl-2 and cellular inhibitor of apoptosis proteins 2, while preventing activation of caspase-3 by cleavage. Furthermore, IGF-II and high concentrations of insulin also evoke phosphorylation of IGF-IR and, like IGF-I, enhance the expression of the steroidogenic acute regulatory protein by Leydig cells. Inhibition of IGF-IR by picropodophyllin decreases the survival of Leydig cells, both in the presence and absence of IGF-I, demonstrating that signaling via the IGF-IR plays an important role in Leydig cell survival.     

Chronic Alcohol Ingestion Enhances Tumor Necrosis Factor-α Expression and Salivary Gland Apoptosis

We investigated the extent of induction in sublingual salivary gland cells apoptosis and tumor necrosis factor-α (TNF-α) expression with chronic ethanol ingestion. The experiments were conducted on rats pair-fed for 8 weeks with alcohol-containing and control liquid diet. The animals were killed, their sublingual glands dissected, and the glandular tissue used for quantitization of TNF-α expression and the assays of acinar cells apoptosis employing sandwich enzyme immunoassay for histone-associated DNA fragments. The mean value for TNF-α in sublingual gland of the control group was 22.3 pg/mg of protein and showed a 1.6-fold increase in the chronic ethanol diet group to 36.5 pg/mg of protein. In comparison with the controls, the sublingual gland of the chronic ethanol diet group also exhibited a 3.4-fold enhancement in acinar cell apoptosis. These findings suggest that chronic ethanol ingestion causes the enhancement in TNF-α expression and leads to the induction in salivary gland acinar cells apoptosis. Thus, the diminished secretion of saliva in alcoholics may be a direct result of increased salivary gland apoptosis.     

Effect of lycopene on insulin-like growth factor-I,IGF binding protein-3 and IGF type-I receptor in prostate cancer cells

Purpose Prostate cancer is the second most common cancer that leads to death in elderly men. The risk of prostate cancer prevalence is often associated with the elevated level of insulin-like growth factor-I (IGF-I) and decreased level of IGF-binding protein 3 (IGFBP-3). Lycopene, a carotenoid, reduces the proliferation of cancer cells and induces apoptosis. Hence, higher intake of lycopene can be associated with the lower risk of prostate cancer. However, the mechanism of action of lycopene in the prevention of prostate cancer is still unclear. The present study was carried out to study the effects of lycopene on the components of IGF system and apoptosis in androgen-independent prostate cancer cells (PC-3 cells). Methods PC-3 cells were treated with various concentrations of lycopene, (20, 40 and 60 μM) for 24, 48, 72 and 96 h. IGF-I, IGFBP-3 and IGF-I receptor (IGF-IR) levels in lycopene-treated cells were evaluated. Annexin V and propidium iodide (PI) binding studies were done to assess apoptosis. Results PC-3 cells treated with lycopene showed a significant decrease in cell proliferation. Lycopene, at a dose of 40 μM, significantly increased the level of IGFBP-3. Lycopene-induced apoptosis was confirmed by annexin V and PI binding. Lycopene-induced DNA fragmentation was absent after 24 h treatment whereas the same was observed after 48 h treatment. There was a significant decrease in the IGF-IR expression after the cells were treated with lycopene and IGF-I. Conclusion The data obtained suggest that the components of the IGF system may act as a positive regulator of lycopene-induced apoptosis in PC-3 cells. Thus, the observed lycopene-induced biological effects and their associated mechanisms are encouraging and may lead to the development of a highly successful drug for the treatment of prostate cancer.     

Inhibition of insulin-like growth factor-I signaling by ethanol in neuronal cells

BACKGROUND: Ethanol inhibits insulin-like growth factor-I receptor (IGF-IR) activation. However, the potency of ethanol for inhibition of the IGF-IR and other receptor tyrosine kinases varies considerably among different cell types. We investigated the effect of ethanol on IGF-I signaling in several neuronal cell types. METHODS: IGF-I signaling was examined in SH-SY5Y neuroblastoma cells, primary cultured rat cerebellar granule neurons, and rat NG-108 neuroblastoma x glioma hybrids. The tyrosine phosphorylation of IGF-IR, IRS-2, Shc, and p42/p44 MAP kinase (MAPK), and the association of Grb-2 with Shc, were examined by immunoprecipitations and Western blotting. RESULTS: IGF-I-mediated tyrosine phosphorylation of MAPK was inhibited by ethanol in all cell lines. IGF-IR autophosphorylation was markedly inhibited by ethanol in SH-SY5Y cells, was only mildly inhibited in cerebellar granule neurons, and was unaffected in rat NG-108 cells. In vitro tyrosine autophosphorylation of immunopurified IGF-IR obtained from all cell lines was inhibited by ethanol. There was also differential ethanol sensitivity of IRS-2 and Shc phosphorylation, and the association of Shc with IRS-2, among the different cell types. CONCLUSIONS: The findings demonstrate that IGF-I-mediated MAPK activation is a sensitive target of ethanol in diverse neuronal cell types. The data are consistent with ethanol-induced inhibition of IGF-IR activity, although the extent of IGF-IR tyrosine autophosphorylation per se is a poor marker of the inhibitory action of ethanol on this receptor. Furthermore, despite uniform inhibition of MAPK in the different neuronal cell types, tyrosine phosphorylation of proximal mediators of the IGF-IR are differentially inhibited by ethanol.     

Targeted disruption of the IGF-I receptor gene decreases cellular proliferation in mammary terminal end buds

IGF-I mediates mammary ductal development through stimulation of terminal end bud (TEB) development; however, no published data exist on the mechanism through which this occurs. The mechanism of IGF-I action on the TEB was studied by determining the requirement for the IGF-I receptor (IGF-IR) in IGF-I-dependent ductal development. We hypothesized that loss of the IGF-IR would disrupt mammary ductal development through a combination of decreased proliferation or increased apoptosis. Because IGF-IR null mice die at birth, embryonic mammary gland transplantation was used to study the effects of a disrupted IGF-IR gene. Analyses of grafts after 4 or 8 wk of development demonstrated a limited growth potential of the null mammary epithelium in virgin hosts. Bromodeoxyuridine labeling and terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling showed that cell proliferation was significantly decreased in null TEBs, but apoptosis was not. In addition, both the size and number of TEBs were reduced in null outgrowths. In pregnant hosts, null ductal growth was stimulated beyond the level seen in virgin hosts. These findings directly establish a proliferation-dependent role for the IGF-IR in the cells of the TEB. Additionally, this study indicates that pregnancy-dependent compensatory mechanisms can stimulate mammary development in the absence of an IGF-IR.     

Effect of Alcohol on the Secretion of Tumor Necrosis Factor-α by Macrophages in the Presence of Rat Serum

Background It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease. Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol. To determine the pathophysiological roles of macrophages in alcoholic liver disease, we examined the effect of ethanol on TNF-α secretion of rat Kupffer cells, alveolar macrophages, and peritoneal macrophages in the presence or absence of LBP.
Methods Kupffer cells, alveolar macrophages, and peritoneal macrophages were isolated from male Sprague Dawley rats. After the preculture in the medium containing 0, 10, 50, and 100 mmol/liter of ethanol, TNF-α secretion by these cells incubated with 100 ng/ml of endotoxin in the presence or absence of LBP (1% rat serum) was determined.
Results In the absence of LBP, an addition of ethanol to the medium suppressed TNF-α secretion of alveolar macrophages. Kupffer cells and peritoneal macrophages were less affected. Addition of LBP led to marked enhancement (7- to 24-fold) of TNF-α secretion of macrophages either with or without ethanol in the medium. Although ethanol tended to suppress TNF-α secretion of these cells, alveolar macrophages were less affected in the presence of LBP.
Conclusions Serum LBP enhances the secretion of TNF-α by macrophages. Alveolar macrophages may be important for excessive production of TNF-α in chronic alcoholics with endotoxemia.     

Spatial distribution of growth hormone receptor, insulin-like growth factor-I receptor and apoptotic chondrocytes during growth plate development

Linear bone growth depends upon proliferation, maturation, and apoptosis of growth plate chondrocytes, processes regulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I). To investigate the contribution of GH, IGF-I and apoptosis to growth plate function, the expression of GH receptor (GHR) and IGF-I receptor (IGF-IR) mRNA were evaluated by in situ hybridization in fractionated costochondral growth plates of growing rats (at 2, 4, and 7 weeks). Apoptosis was determined by TUNEL assay and morphology in histological sections. GHR mRNA was greatest in resting cells with hypertropic cells increasing GHR expression with increasing age. Hypertropic and resting cell IGF-IR mRNA declined over the ages studied. Receptor mRNA expression was altered by exposing cells to GH or IGF-I. GH and IGF significantly decreased GHR mRNA in proliferative cells. GH and IGF also decreased IGF-IR mRNA in resting cells and the 2- and 4-week-old proliferative and hypertropic cells. Treating cells in culture with GH increased the number of apoptotic cells across all ages and zones. Histologically, apoptotic cells were observed at the chondro-osseous junction and within actively proliferating chondrocytes but not in resting cells. Apoptosis was highest at 4 weeks of age with lateral regions displaying the greatest number of cells undergoing apoptosis. These data indicate that apoptosis plays a role in growth plate function, particularly spatial configuration as indicated by the preferential lateral cell apoptosis. The susceptibility of proliferative cells to GHR and IGF-IR down regulation during the period of greatest apoptosis supports a role for the GH-IGF axis in both proliferation and apoptosis during growth plate development.     

Effect of cholesterol depletion on mitogenesis and survival: the role of caveolar and noncaveolar domains in insulin-like growth factor-mediated cellular function

The type 1 IGF receptor (IGF-IR) is thought to localize to a subset of lipid rafts, known as caveolae, but the impact on IGF signaling remains controversial. We investigated this potential regulatory mechanism by assessing IGF function in caveolae-positive (3T3L1 and NWTb3) and -negative (HepG2) cells. Coimmunoprecipitation studies demonstrated that IGF-IR and insulin receptor substrate 1 associated with caveolin, a caveolar marker, in 3T3L1 and NWTb3 cells. Subcellular fractionation showed that methyl-cyclodextrin, which disrupts lipid rafts by sequestration of cholesterol, disrupted the colocalization of caveolin and the IGF-IR at the plasma membrane. Methyl-cyclodextrin did not alter IGF-I-induced 3T3L1 or NWTb3 proliferation but significantly impaired the ability of IGF-I to protect these cells from apoptosis. Immunoblotting revealed that methyl-cyclodextrin had no effect on IGF-I-induced activation of the IGF-IR or insulin receptor substrate 1 but increased and decreased the phosphorylation of MAPK and protein kinase B, respectively. In caveolae-negative HepG2 cells, the effect of methyl-cyclodextrin on IGF signaling and cellular function was similar to that observed in caveolae-positive 3T3L1 and NWTb3 cells. Furthermore, transfecting caveolin into HepG2 cells to give morphologically identifiable caveolae made no difference to IGF action, despite a demonstrable interaction between caveolin and the IGF-IR. This suggests that although IGF-IR localizes to caveolin-rich subcellular fractions and coimmunoprecipitates with caveolin, caveolae may not be obligatory for IGF signaling.     

A familial insulin-like growth factor-I receptor mutant leads to short stature: clinical and biochemical characterization

CONTEXT: IGF-I/IGF-I receptor (IGF-IR) signaling pathways play important roles in longitudinal growth. A novel Arg481Glu (R481Q) mutation in IGF-IR was detected in a family with intrauterine and postnatal growth retardation. OBJECTIVE: The objective of the study was to explore the mechanism whereby the R481Q mutation may be causative in growth retardation. PATIENTS: A 13-yr-old girl with short stature was studied for functional analysis of the R481Q mutation in the IGF-IR. RESULTS: Two members of a family who showed intrauterine and postnatal growth retardation, with increased serum IGF-I levels, demonstrated a substitution of arginine for glutamine at 481 (R481Q) in the IGF-IR. This mutation results in the formation of an altered fibronectin type III domain within the alpha-subunit. NIH-3T3 fibroblasts that overexpress the human wild-type or R481Q mutant IGF-IR demonstrated normal cell surface ligand binding by 125I-IGF-I binding assay. However, the fold increase of IGF-I stimulated tyrosine phosphorylation of the IGF-IR beta-subunit as well as downstream activation of ERK1/2 and Akt was reduced in cells overexpressing the mutant receptor. Additionally, basal and IGF-I-stimulated levels of cell proliferation were also reduced in cells overexpressing the mutant receptor. CONCLUSION: Our results demonstrate that NIH-3T3 cells overexpressing a mutant form of the Igf1r gene, in which arginine at 481 is substituted by glutamine, lead to reduced levels of the fold increase of IGF-IR beta-subunit phosphorylation as well as ERK1/2 and Akt phosphorylation and was accompanied by decreased cell proliferation. These results are postulated to be the cause of intrauterine and postnatal growth retardation in the described patients.     

Increased, not decreased activation of the insulin-like growth factor (IGF) receptor signalling pathway during ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro. The IGF-I receptor (IGF-IR) is believed to mediate protective effects of the IGFs against apoptosis. To determine whether ceramide-mediated induction of apoptosis involved a decreased survival effect of the IGF-IR, apoptosis was induced in IGF-I receptor positive (R+) and negative (R-) murine fibroblasts by incubation with increasing doses of the sphingolipid analogue, C2 ceramide. Lower ceramide doses were required to induce death in receptor negative compared with receptor positive fibroblasts (P< 0.05 at ceramide doses of 2 microM or greater), not only corroborating evidence that the IGF-I receptor functions as a survival receptor, but also suggesting that ceramide is not inducing apoptosis by suppressing a survival effect of the IGF-IR. Ceramide has been reported to induce death through suppression of MAP kinase, and activation of JUN kinase signalling; since our initial data suggested that ceramide had not affected an anti-apoptotic signalling event of the IGF-IR, we monitored the activation of these enzymes. To our surprise, in the presence of ceramide, not only was JUN kinase activity increased, but so too was MAP kinase. Inhibition of MAP kinase, using the MEKK inhibitor, PD98059, significantly reduced ceramide-induced cell death (P< 0. 001). Ceramide also enhanced IGF-induced tyrosine phosphorylation of the IGF-I receptor and activated PI-3 kinase. The cumulative effects of these events resulted in increased progression to the G2 phase of the cell cycle, arrest without subsequent mitosis, and apoptosis. These results indicate that ceramide is capable of eliciting apparently contradictory events within a single cell type, and suggest that in the presence of an IGF-IR, survival is enhanced because ceramide can activate PI-3 kinase, believed to be an anti-apoptotic enzyme.     

Interactions between IGF-I, estrogen receptor-α (ERα), and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells

Understanding of the interactions between estradiol (E?) and IGF-I is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating noninterfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions, and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human GH plus epidermal growth factor, but E? did not cause an increase in the number of the IGF-IR.low cells compared to controls. The proliferation rate of IGF-IR.low cells was only reduced in response to E? compared to controls, whereas their basal and hormone-stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E?, without affecting control cells. Furthermore, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. In conclusion, suppressing IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK, which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate.     

Effects of glycyrrhizin on immune-mediated cytotoxicity

Intravenous administration of glycyrrhizin is known to decrease elevated plasma transaminase levels in patients with chronic viral hepatitis, in which immune-mediated cytotoxicity by cytotoxic T lymphocytes and tumour necrosis factor (TNF)-α is considered to play an important pathogenic role. However, the immunological interpretation of the transaminase-lowering action of glycyrrhizin is not known. Studies were performed to elucidate this action immunologically by assessing the effects of glycyrrhizin on immune-mediated cytotoxicity using an antigen-specific murine CD4⁺ T hybridoma line, which exhibits cytotoxicity against antigen-presenting cells after stimulation with specific antigen, and a murine TNF-α-sensitive fibroblast line. Glycyrrhizin inhibited the cytotoxic activity of the T cells against antigen-presenting cells and also suppressed TNF-α-induced cytotoxicity in the TNF-α-sensitive cell line in vitro. These results suggest that the decrease of elevated transaminase levels by glycyrrhizin in patients with chronic viral hepatitis is mediated in part by inhibition of immune-mediated cytotoxicity against hepatocytes.     

Na+/H+ exchanger mediates TNF-α-induced hepatocyte apoptosis via the calpain-dependent degradation of Bcl-xL

Background and Aim:  It is well known that tumor necrosis factor-α (TNF-α) induces hepatocyte apoptosis and contributes to liver diseases. However, the exact mechanisms are not well understood.
Methods:  In the present study, we reported that Na⁺/H⁺ exchanger (NHE) is involved in TNF-α-induced hepatocyte apoptosis.
Results:  TNF-α time dependently induced an increase in NHE activity in hepatocytes, but cariporide, an NHE inhibitor, blocked the TNF-α-induced increase of NHE activity in a dose-dependent manner. Increased NHE activity induced by TNF-α was associated with increased intracellular calcium (Ca²⁺_i) concentration and calpain activity. Cariporide reversed these effects induced by TNF-α. In addition, TNF-α downregulated Bcl-xL, an anti-apoptotic protein, but not mRNA levels. The inhibition of either calpain or NHE blocked the TNF-α-induced decrease of the Bcl-xL protein. TNF-α did not change the pro-apoptotic Bax and Bak protein levels. Cariporide, calcium remover 1,2-bis (2-aminophenoxy) ethane-N,N,N0,N0–tetraacetic acid, or calpain inhibitor benzyloxycarbonyl-leucyl-leucinal attenuated TNF-α-induced hepatocyte apoptosis.
Conclusion:  TNF-α via NHE results in hepatocyte apoptosis through the calcium/calpain/Bcl-xL pathway.     

Hepatitis C virus F protein inhibits cell apoptosis by activation of intracellular NF-κB pathway

Aim:  To observe the influence of HCV F protein on apoptosis of HepG2 cells, and explore the association between F protein and NF-κB signal pathway.
Methods:  HCV 1b F gene containing HepG2-F cells and HCV 1b C gene containing HepG2-C cells were treated with 100 IU/mL TNF-α, and analyzed by flow cytometry, Western blotting, and dual luciferase reporter assay. Empty plasmid pcDNA3.1⁺ containing HepG2-3.1 cells were used as control.
Results:  (i) With the treatment of TNF-α for 18 h, the apoptosis rates (AR) of HepG2-F and HepG2-3.1 cells were 0.41% (± 0.11%) and 37.43% (± 2.03%) respectively, while that of HepG2-C was 4.07% (± 0.18%). At 36 h after TNF-α treatment, the AR of HepG2-F and HepG2-3.1 cells were 10.03% (± 0.41%) and 44.63% (± 3.37%), and that of HepG2-C was 14.95% (± 0.85%). (ii) After the treatment of TNF-α for 0.5–18 h, the p65 contents in the whole cells of HepG2-F and HepG2-3.1 showed no significant difference ( P  = 0.34, t  = 1.08), while the p65 contents in the nucleus of HepG2-F and HepG2-3.1 cells were 3.8–1.9 times and 1.8–1.0 times higher than that in the non-treated cells ( P  = 0.013, t  = 4.25). (iii) The relative luciferase unit (RLU) of the HepG2 cells, co-transfected with pcDNA3.1-F and pNF-κB-luc, and then treated with TNF-α (100 IU/mL) for 18 h, showed a pcDNA3.1-F dose-dependent increase.
Conclusion:  HCV F protein can over-activate NF-κB signal pathway, which makes HepG2-F cells able to resist TNF-α induced apoptosis.     

Glucagon-like peptide-2 activates beta-catenin signaling in the mouse intestinal crypt: role of insulin-like growth factor-I

Chronic administration of glucagon-like peptide-2 (GLP-2) induces intestinal growth and crypt cell proliferation through an indirect mechanism requiring IGF-I. However, the intracellular pathways through which IGF-I mediates GLP-2-induced epithelial tropic signaling remain undefined. Because beta-catenin and Akt are important regulators of crypt cell proliferation, we hypothesized that GLP-2 activates these signaling pathways through an IGF-I-dependent mechanism. In this study, fasted mice were administered Gly(2)-GLP-2 or LR(3)-IGF-I (positive control) for 0.5-4 h. Nuclear translocation of beta-catenin in non-Paneth crypt cells was assessed by immunohistochemistry and expression of its downstream proliferative markers, c-myc and Sox9, by quantitative RT-PCR. Akt phosphorylation and activation of its targets, glycogen synthase kinase-3beta and caspase-3, were determined by Western blot. IGF-I receptor (IGF-IR) and IGF-I signaling were blocked by preadministration of NVP-AEW541 and through the use of IGF-I knockout mice, respectively. We found that GLP-2 increased beta-catenin nuclear translocation in non-Paneth crypt cells by 72 +/- 17% (P < 0.05) and increased mucosal c-myc and Sox9 mRNA expression by 90 +/- 20 and 376 +/- 170%, respectively (P < 0.05-0.01), with similar results observed with IGF-I. This effect of GLP-2 was prevented by blocking the IGF-IR as well as ablation of IGF-I signaling. GLP-2 also produced a time- and dose-dependent activation of Akt in the intestinal mucosa (P < 0.01), most notably in the epithelium. This action was reduced by IGF-IR inhibition but not IGF-I knockout. We concluded that acute administration of GLP-2 activates beta-catenin and proliferative signaling in non-Paneth murine intestinal crypt cells as well as Akt signaling in the mucosa. However, IGF-I is required only for the GLP-2-induced alterations in beta-catenin.     

Moderately High Consumption of Ethanol Suppresses Bone Resorption in Ovariectomized but not in Sexually Intact Adult Female Rats

Epidemiological studies suggest that moderate consumption of alcoholic beverages may be beneficial for bone in postmenopausal women. To investigate prospectively these uncontrolled obsewations, female rats were divided in four groups of 10 animals each and treated with 1) ovariectomy (OVX) and 2.5% ethanol diet (OVX-ETOH group), 2) OVX and control diet (OVX-C group), 3) sham surgery and 25% ethanol diet (SHAM-ETOH group), or 3) sham surgery and control diet (SHAM-C group). Three weeks after surgery, bone histomor-phometfy revealed that the OVX-C group, as expected, had lower trabecular bone volume and higher parameters of bone formation and resorption than the SHAM-C group (p < 0.01). Intake of ethanol did not change these parameters in the SHAM rats, but in the OVX rats it was associated with sharp reduction in parameters of bone resorption (p < 0.01) without a concomitant effect on parameters of bone formation. The cytokines are believed to contribute to accelerated bone resorption during the early postmenopausal period. Indeed, the peripheral blood monocybc cells (PBMC) from the OVX-C rats produced higher amounts of TNF-α than the PBMC from the SHAM-C rats (p < 0.05) and administration of ethanol prevented this increase in OVX rats but had no effect in SHAM rats. In summary, short-tetm intake of moderate doses of ethanol was associated with markedly different eftects in rats with and without ovarian function. Although ethanol had no significant effect on the bone tissue and TNF-α production of the SHAM rats, it was associated with markedly lower parameters of bone resorption and less TNF-α production in the OVX animals. This suggests that exposure to low-dose ethanol may protect from osteopenia following cessation of ovarian function.     

The Production of Tumor Necrosis Factor-α by Macrophages in Rats With Acute Alcohol Loading

Background: It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease. Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol. To study the pathophysiological roles of macrophages in alcoholic liver diseases, we examined the production of TNF-α by rat Kupffer cells, splenic macrophages, and alveolar macrophages with acute alcohol loading in the presence or absence of LBP.
Methods: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from male Wistar rats given 5 mg/g body weight of ethanol intraperitoneally after an hour. The production of TNF-α by these cells incubated with endotoxin 100 ng/ml in the presence or absence of LBP (1% rat serum) was determined.
Results: Acute alcohol loading did not affect the production of TNF-α by Kupffer cells. With acute alcohol loading, splenic macrophages tended to produce more TNF-α. Alveolar macrophages produced more TNF-α than Kupffer cells, and although the production of TNF-α by alveolar macrophages tended to be suppressed by acute alcohol loading, the production of TNF-α by alveolar macrophages still remained high in the presence of rat serum.
Conclusions: Splenic macrophages and alveolar macrophages may be related to excessive production of TNF-α in acute alcoholics with endotoxemia.