RESUMO
Morbid obesity is considered a systemic inflammatory state. The objective of this project was to characterize the adipokine, cytokine and chemokine protein profile in serum from control, lean and obese mice. We hypothesized that chemokines and cytokines are altered by caloric restriction and diet-induced obesity as a function of changes in body composition. Six-week-old female C57BL/6N mice (n = 12 per group) were randomized to one of three diets: control (fed ad libitum); lean (30% calorie-restricted regimen relative to control) and diet-induced obese (DIO; high calorie diet, fed ad libitum). Body weight, body composition and food intake were monitored throughout the study. After 10 weeks on the diets, blood samples were collected, and adipokine/cytokine/chemokine serum profiles were measured by antibody array. Lean mice, relative to the control group, displayed increased concentrations of insulin-like growth factor (IGF) binding protein-3, -5 and -6 and adiponectin and decreased IGF-1. These mice also showed increased concentrations of interleukin (IL)-10, IL-12 p40/p70, eotaxin, monocyte chemoattractant protein-5 and SDF-1. In contrast, DIO mice displayed increased leptin, IL-6 and LPS-induced chemokine and decreased concentrations of all chemokines/cytokines measured relative to control mice. As such, these data indicate that DIO may lead to an inflammatory state characterized as a shift towards a T helper lymphocyte type 1-skewed responsiveness. The demonstration of differential adipokine, cytokine and chemokine protein profile in control, lean and DIO mice may have implications for immune responsiveness and risk of disease.
Assuntos
Citocinas/sangue , Dieta/efeitos adversos , Mediadores da Inflamação/sangue , Obesidade/sangue , Adipocinas/sangue , Adiposidade/fisiologia , Animais , Composição Corporal/fisiologia , Peso Corporal/fisiologia , Quimiocinas/sangue , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Análise Serial de Proteínas/métodos , Distribuição Aleatória , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: We tested the relative importance of a low-glycemic response versus a high glycemic response breakfast meal on postprandial serum glucose, insulin and free fatty acid (FFA) responses after consumption of a standardized mid-day meal in adult individuals with Type 2 diabetes mellitus (DM). DESIGN: Following an overnight fast of 8-10 h, a randomized crossover intervention using control and test meals was conducted over a 3-week-period. A fasting baseline measurement and postprandial measurements at various time intervals after the breakfast and mid-day meal were taken. SUBJECTS: Forty-five Type 2 DM subjects completed the requirements and were included in the study results. INTERVENTIONS: Two different breakfast meals were administered during the intervention: (A) a high glycemic load breakfast meal consisting of farina (kJ 1833; carbohydrate (CHO) 78 g and psylium soluble fiber 0 g), (B) a low-glycemic load breakfast meal consisting of a fiber-loop cereal (kJ 1515; CHO 62 g and psyllium soluble fiber 6.6 g). A standardized lunch was provided approximately 4 h after breakfast. Blood plasma concentrations and area under the curve (AUC) values for glucose, insulin and FFA were measured in response to the breakfast and mid-day lunch. Statistical analyses were performed using SAS software (8.02). Comparisons between diets were based on adjusted Bonferroni t-tests. RESULTS: In post-breakfast analyses, Breakfast B had significantly lower area under the curve (AUC) values for plasma glucose and insulin compared to Breakfast A (P<0.05) (95% confidence level). The AUC values for FFA were higher for Breakfast B than for Breakfast A (P<0.05) (95% confidence level). Post-lunch analyses indicated similar glucose responses for the two breakfast types. Insulin AUC values for Breakfasts B were significantly lower than Breakfast A (P<0.05) (95% confidence level). The AUC values for FFA were unaffected by breakfast type. CONCLUSIONS: These data indicate that ingesting a low-glycemic load meal containing psyllium soluble fiber at breakfast significantly improves the breakfast postprandial glycemic, insulinemic and FFA responses in adults with Type 2 DM. These data revealed no residual postprandial effect of the psyllium soluble fiber breakfast meal beyond the second meal consumed. Thus, there was no evidence of an improvement postprandially in the glycemic, insulinemic and FFA responses after the consumption of the lunch meal.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético/fisiologia , Ácidos Graxos não Esterificados/sangue , Índice Glicêmico , Insulina/metabolismo , Adulto , Idoso , Área Sob a Curva , Estudos Cross-Over , Diabetes Mellitus Tipo 2/sangue , Dieta , Carboidratos da Dieta/administração & dosagem , Carboidratos da Dieta/metabolismo , Fibras na Dieta/administração & dosagem , Fibras na Dieta/metabolismo , Jejum , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Prandial , Fatores de TempoRESUMO
Increased cytoplasmic beta-catenin levels and the associated nuclear beta-catenin/T-cell factor (Tcf)-lymphoid enhancer factor (LEF) complex formation have been frequently found in colon cancer. In this context, overproduction of nitric oxide (NO) attributable to inflammatory stimuli in diseases such as ulcerative colitis and Crohn's disease may-contribute to colonic carcinogenesis. Therefore, we examined the modulation by NO of cytoplasmic beta-catenin levels and the formation of the nuclear beta-catenin/LEF-1 DNA binding complex in conditionally immortalized mouse colonic epithelial cells that differed in adenomatous polyposis coli (Apc) genotype, namely young adult mouse colon (YAMC; Apc+/+) and immortal mouse colon epithelium (IMCE; ApcMin/+). Unlike most colon cancer cell lines, this pair of cell lines has either nondetectable or low basal level of beta-catenin when they are cultured under nonpermissive and nonproliferative conditions. Using electrophoretic mobility shift assays, we found that NO-releasing agents (E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide and S-nitroso-N-acetylpenicillamine greatly enhanced the formation of beta-catenin/LEF-1 DNA binding complex in a concentration- and time-dependent fashion in YAMC and IMCE cells. Significantly, IMCE cells showed a markedly greater amount of nuclear beta-catenin/LEF-1 DNA binding complex in response to NO. Super shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Western blot analysis of the soluble cytoplasmic fractions demonstrated that these NO donors caused differential accumulation of cytoplasmic beta-catenin in YAMC and IMCE. In conclusion, this study indicates that the defective beta-catenin degradation machinery attributable to ApcMin/+ mutation in IMCE cells not only affects basal levels but also contributes to NO-induced dysregulation of cytoplasmic beta-catenin and nuclear beta-catenin/LEF-1 DNA binding complex formation.
Assuntos
Colo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes APC , Mucosa Intestinal/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/fisiologia , Nitrocompostos/farmacologia , Penicilamina/análogos & derivados , Transativadores , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Colo/efeitos dos fármacos , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Genótipo , Mucosa Intestinal/efeitos dos fármacos , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Penicilamina/farmacologia , S-Nitroso-N-Acetilpenicilamina , Fatores de Transcrição/isolamento & purificação , beta CateninaRESUMO
The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic helix-loop-helix transcription factor that heterodimerizes with the aryl hydrocarbon receptor to mediate signal transduction pathways inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and other planar aromatic hydrocarbons. Monoclonal antibodies (MAbs) have been raised against a carboxyl-terminal 19-amino acid peptide hapten (MAb 2B10) and against a carboxyl-terminal 378-amino acid polypeptide-staphylococcal Protein A fusion protein (MAb 4G9) of Arnt and their characterization is described. Western blot experiments show that both MAbs specifically cross-react with an approximately 85-kDa band in cytosol prepared from COS-7 cells transfected with the full length human Arnt cDNA pBMSNeo-D24-1 and in Hepa 1c1c7 cytosol but not in Arnt-deficient Hepa 1-C4 mutant cytosol. Velocity sedimentation of Hepa 1c1c7 cytosol on sucrose gradients and Superose 6 gel permeation chromatography were used to estimate the sedimentation coefficient. Stokes radius, and relative molecular mass of Arnt as approximately 3.6-4.1 S, 6.8 nm, and 101-115 kDa, respectively. These results indicate that Arnt probably exists in monomeric form in Hepa 1c1c7 cytosolic extracts. Laser scanning confocal microscopy and indirect immunofluorescence microscopy revealed Arnt to be distributed throughout the non-nucleolar portion of the nucleus of Hepa 1c1c7, VT(2) (Hepa 1-C4T mutant cell line deficient in Arnt function and stably transfected with pBMSNeo D24-1, expressing the full length human Arnt cDNA), and HeLa cells. The establishment of the nuclear localization of Arnt in human and murine cell lines shown here indicates that its nuclear localization may be conserved across species. Immunofluorescence analysis of Arnt in three cell lines using two MAbs (to distinct epitopes) provides evidence that suggests that the aryl hydrocarbon receptor heterodimerizes with Arnt in the nucleus.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Ligação a DNA , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição/imunologia , Sequência de Aminoácidos , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Linhagem Celular , Epitopos/imunologia , Feminino , Sequências Hélice-Alça-Hélice , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Absorptiometric, histomorphometric, and chemical analyses of bones from growing rats fed diets with low (0.2%, w/w), marginal (0.4%, w/w), or adequate (0.8%, w/w) calcium (Ca) content with or without phytate were compared. Phytate was added to each diet in a molar ratio of 19:1 to calcium. Male weanling Sprague-Dawley rats were fed one of the six diets for 8 weeks. At the end of 8 weeks, rats were killed, and mandibles, femurs, and tibias were removed. Bone density profiles were determined on the mandibles and femurs using single photon absorptiometry. Femurs were also used for calcium and phosphorus analyses. Tibias were used for histomorphometric analyses. Bone density of the femurs and mandibles increased as dietary Ca increased. The only effect of phytate addition measured was in the 0.8% calcium diet, where density was lower in rats fed the phytate-containing 0.8% calcium diet. Femur calcium concentration also increased as dietary Ca increased and was unaffected by addition of phytate. Femur phosphorus concentration was unaffected by dietary Ca levels but was increased by 10% when phytate was added to the diet. Bone density values were highly correlated with bone calcium and phosphorus levels (r = 0.94). Rats fed the 0.2% calcium diets had 20% lower mineralized bone area and 20% larger medullary cavity area than rats fed the other diets. Bone densitometry appears to be useful for determining changes in bone occurring in growing rats fed low, marginal, and adequate levels of dietary Ca. Bone density values also correlated well with chemically determined calcium and phosphorus concentrations and with histomorphometric data.
Assuntos
Osso e Ossos/química , Cálcio da Dieta/administração & dosagem , Ácido Fítico/administração & dosagem , Absorciometria de Fóton , Animais , Peso Corporal , Densidade Óssea , Osso e Ossos/anatomia & histologia , Cálcio/análise , Fêmur/anatomia & histologia , Fêmur/química , Masculino , Mandíbula/anatomia & histologia , Mandíbula/química , Fósforo/análise , Ratos , Ratos Endogâmicos , Tíbia/anatomia & histologia , Tíbia/químicaRESUMO
The hepatitis B virus X-associated protein 2 (XAP2) is an immunophilin homolog and core component of the aryl hydrocarbon receptor (AhR). Immunophilins are components of many steroid receptor complexes, serving a largely unknown function. Transiently expressed AhR.YFP (yellow fluorescent protein) localized to the nuclei of COS-1 and NIH-3T3 cells. Co-expression of AhR.YFP with XAP2 restored cytoplasmic localization, which was reversed by 2,3,7, 8-tetrachlorodibenzo-p-dioxin treatment (TCDD). The effect of XAP2 on AhR localization was specific involving a nuclear localization signal-mediated pathway. Examination of the ratio of AhR to XAP2 in the AhR complex revealed that approximately 25% of transiently expressed AhR was associated with XAP2, in contrast with approximately 100% when the AhR and XAP2 were co-expressed. Strikingly, TCDD did not influence these ratios, suggesting that ligand binding initiates nuclear translocation prior to complex dissociation. Analysis of endogenous AhR in Hepa-1 cells revealed that approximately 40% of the AhR complex was associated with XAP2, predicting observed AhR localization to cytoplasm and nuclei. This study reveals a novel functional role for the immunophilin-like component of a soluble receptor complex and provides new insight into the mechanism of AhR-mediated signal transduction, demonstrating the existence of two structurally distinct and possibly functionally unique forms of the AhR.
Assuntos
Proteínas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Frações Subcelulares/metabolismo , Células 3T3 , Animais , Células COS , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The aryl hydrocarbon receptor (AhR) is known to mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxic effects. Immunocytochemical studies revealed that AhR in HeLa cells is localized throughout the cell. Upon TCDD treatment most of the cytoplasmic receptor is translocated into the nucleus in a time-dependent manner. A significant amount of AhR was found to be tightly associated with the nuclear fraction of untreated HeLa cells. The level of receptor in the nuclear fraction was approximately 16% of the total cellular receptor pool. Further characterization of AhR heterocomplex from the HeLa nuclear fraction by sucrose density gradient analysis revealed that the AhR was present in the 6 S form, and that the nuclear AhR could be coimmunoprecipitated using anti-Arnt mAb. The ability of the AhR to specifically interact with dioxin-responsive elements (DRE) was demonstrated utilizing wild-type and two mutant DREs in gel shift assays. These results would suggest that, in HeLa cells, the AhR-Arnt heterodimer is associated with the nuclear fraction under normal culture conditions. Therefore, HeLa cells can be used as a model system to study the biochemical and molecular function of the Ah receptor and the process that leads to activation of the AhR in the absence of exogenous ligand.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Receptores de Hidrocarboneto Arílico/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto , Sequência de Bases , Núcleo Celular/efeitos dos fármacos , Células HeLa , Humanos , Ligantes , Dados de Sequência Molecular , Dibenzodioxinas Policloradas/farmacologia , Testes de Precipitina , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.
Assuntos
Enterócitos/enzimologia , Isoenzimas/biossíntese , Óxido Nítrico/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Transativadores , Proteína da Polipose Adenomatosa do Colo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ciclo-Oxigenase 2 , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Indução Enzimática/efeitos dos fármacos , Sequestradores de Radicais Livres/metabolismo , Sequestradores de Radicais Livres/farmacologia , Interferon gama/farmacologia , Isoenzimas/genética , Lipopolissacarídeos/farmacologia , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/antagonistas & inibidores , Doadores de Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Penicilamina/análogos & derivados , Penicilamina/antagonistas & inibidores , Penicilamina/metabolismo , Penicilamina/farmacologia , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade/efeitos dos fármacos , Fatores de Transcrição/metabolismo , beta CateninaRESUMO
Mutations in Apc underlie the intestinal lesions in familial adenomatous polyposis and are found in >85% of sporadic colon cancers. They are frequently associated with overexpression of prostaglandin endoperoxide H synthase-2 (PGHS-2) in colonic adenomas. It has been suggested that Apc mutations are linked mechanistically to increased PGHS-2 expression by elevated nuclear accumulation of beta-catenin-Tcf-LEF transcription complex. In the present study, we show that PGHS-2 is differentially expressed in mouse colonic epithelial cells with distinct Apc status. Cells with a mutated Apc expressed markedly higher levels of PGHS-2 mRNA and protein and produced significantly more prostaglandin E2 than cells with normal Apc. Using electrophoretic mobility shift assays, we demonstrate that DNA-beta-catenin-LEF-1 complex formation is differentially induced in these two cell lines in an Apc-dependent manner. Our data indicate that the differential induction of beta-catenin-LEF-1 complex correlates closely with differential expression of PGHS-2. These findings support the hypothesis that the differential expression of PGHS-2 is mediated through the proposed beta-catenin/Tcf-LEF signaling pathway.