RESUMO
SCOPE: This study investigates the mechanism of action and functional effects of coffee extracts in colonic cells, on intestinal stem cell growth, and inhibition of dextran sodium sulfate (DSS)-induced intestinal barrier damage in mice. METHODS AND RESULTS: Aqueous coffee extracts induced Ah receptor (AhR) -responsive CYP1A1, CYP1B1, and UGT1A1 gene expression in colon-derived Caco2 and YAMC cells. Tissue-specific AhR knockout (AhRf/f x Lgr5-GFP-CreERT2 x Villin-Cre), wild-type (Lgr5-CreERT2 x Villin-Cre) mice are sources of stem cell enriched organoids and both coffee extracts and norharman, an AhR-active component of these extracts inhibited stem cell growth. Coffee extracts also inhibit DSS-induced damage to intestinal barrier function and DSS-induced mucosal inflammatory genes such as IL-6 and TGF-ß1 in wild-type (AhR+/+ ) but not AhR-/- mice. In contrast, coffee does not exhibit protective effects in intestinal-specific AhR knockout mice. Coffee extracts also enhanced overall formation of AhR-active microbial metabolites. CONCLUSIONS: In colon-derived cells and in the mouse intestine, coffee induced several AhR-dependent responses including gene expression, inhibition of intestinal stem cell-enriched organoid growth, and inhibition of DSS-induced intestinal barrier damage. We conclude that the anti-inflammatory effects of coffee in the intestine are due, in part, to activation of AhR signaling.
Assuntos
Café , Colo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Células CACO-2 , Colo/metabolismo , Citocromo P-450 CYP1A1/fisiologia , Citocromo P-450 CYP1B1/fisiologia , Sulfato de Dextrana/toxicidade , Feminino , Humanos , Masculino , CamundongosRESUMO
Proteins are often credited as the macromolecule responsible for performing critical cellular functions, however lipids have recently garnered more attention as our understanding of their role in cell function and human health becomes more apparent. Although cellular membranes are the lipid environment in which many proteins function, it is now apparent that protein and lipid assemblies can be organized to form distinct micro- or nanodomains that facilitate signaling events. Indeed, it is now appreciated that cellular function is partly regulated by the specific spatiotemporal lipid composition of the membrane, down to the nanosecond and nanometer scale. Furthermore, membrane composition is altered during human disease processes such as cancer and obesity. For example, an increased rate of lipid/cholesterol synthesis in cancerous tissues has long been recognized as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids/cholesterol to cellular function in disease models is not yet fully understood. Furthermore, an important consideration in regard to human health is that diet is a major modulator of cell membrane composition. This can occur directly through incorporation of membrane substrates, such as fatty acids, e.g., n-3 polyunsaturated fatty acids (n-3 PUFA) and cholesterol. In this review, we describe scenarios in which changes in membrane composition impact human health. Particular focus is placed on the importance of intrinsic lipid/cholesterol biosynthesis and metabolism and extrinsic dietary modification in cancer and its effect on plasma membrane properties.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Neoplasias/prevenção & controle , Membrana Celular/patologia , Colesterol/química , Dieta , Ácidos Graxos Ômega-3/química , Humanos , Metabolismo dos Lipídeos/genética , Neoplasias/dietoterapia , Neoplasias/metabolismo , Neoplasias/patologia , Obesidade/metabolismo , Obesidade/patologiaRESUMO
Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/ultraestrutura , Membrana Celular/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Idoso , Idoso de 80 Anos ou mais , Membrana Celular/química , Membrana Celular/fisiologia , Gorduras na Dieta/administração & dosagem , Suplementos Nutricionais , Ácido Eicosapentaenoico/sangue , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/sangue , Feminino , Óleos de Peixe/administração & dosagem , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Lipídeos de Membrana/sangue , Lipídeos de Membrana/química , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfolipídeos/sangue , Fosfolipídeos/química , Projetos PilotoRESUMO
We have demonstrated that the combination of bioactive components generated by fish oil (containing n-3 polyunsaturated fatty acids) and fermentable fiber (leading to butyrate production) act coordinately to protect against colon cancer. This is, in part, the result of an enhancement of apoptosis at the base of the crypt across all stages (initiation, promotion, and progression) of colon tumorigenesis. As mitochondria are key organelles capable of regulating the intrinsic apoptotic pathway and mediating programmed cell death, we investigated the effects of diet on mitochondrial function by measuring mucosal cardiolipin composition, mitochondrial respiratory parameters, and apoptosis in isolated crypts from the proximal and distal colon. C57BL/6 mice (n=15/treatment) were fed one of two dietary fats (corn oil and fish oil) and two fibers (pectin and cellulose) for 4 weeks in a 2×2 factorial design. In general, diet modulated apoptosis and the mucosal bioenergetic profiles in a site-specific manner. The fish/pectin diet promoted a more proapoptotic phenotype - for example, increased proton leak (Pinteraction=0.002) - compared with corn/cellulose (control) only in the proximal colon. With respect to the composition of cardiolipin, a unique phospholipid localized to the mitochondrial inner membrane where it mediates energy metabolism, fish oil feeding indirectly influenced its molecular species with a combined carbon number of C68 or greater, suggesting compensatory regulation. These data indicate that dietary fat and fiber can interactively modulate the mitochondrial metabolic profile and thereby potentially modulate apoptosis and subsequent colon cancer risk.
Assuntos
Apoptose , Colo/patologia , Neoplasias do Colo/etiologia , Gorduras na Dieta/efeitos adversos , Fibras na Dieta/efeitos adversos , Metabolismo Energético , Mitocôndrias/patologia , Animais , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias do Colo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
The International Agency for Research on Cancer recently released an assessment classifying red and processed meat as "carcinogenic to humans" on the basis of the positive association between increased consumption and risk for colorectal cancer. Diet, however, can also decrease the risk for colorectal cancer and be used as a chemopreventive strategy. Bioactive dietary molecules, such as n-3 polyunsaturated fatty acids, curcumin, and fermentable fiber, have been proposed to exert chemoprotective effects, and their molecular mechanisms have been the focus of research in the dietary/chemoprevention field. Using these bioactives as examples, this review surveys the proposed mechanisms by which they exert their effects, from the nucleus to the cellular membrane. In addition, we discuss emerging technologies involving the culturing of colonic organoids to study the physiological effects of dietary bioactives. Finally, we address future challenges to the field regarding the identification of additional molecular mechanisms and other bioactive dietary molecules that can be utilized in our fight to reduce the incidence of colorectal cancer.
Assuntos
Neoplasias do Colo/prevenção & controle , Dieta Saudável , Regulação da Expressão Gênica , Modelos Biológicos , Nutrigenômica/métodos , Animais , Anticarcinógenos/metabolismo , Anticarcinógenos/uso terapêutico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/microbiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/prevenção & controle , Curcumina/metabolismo , Curcumina/uso terapêutico , Metilação de DNA , Fibras na Dieta/metabolismo , Fibras na Dieta/uso terapêutico , Epigênese Genética , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/uso terapêutico , Fermentação , Microbioma Gastrointestinal , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , MicroRNAs/metabolismo , Nutrigenômica/tendências , Processamento de Proteína Pós-TraducionalRESUMO
A significant increase in cyclooxygenase 2 (COX2) gene expression has been shown to promote cylcooxygenase-dependent colon cancer development. Controversy associated with the role of COX2 inhibitors indicates that additional work is needed to elucidate the effects of arachidonic acid (AA)-derived (cyclooxygenase and lipoxygenase) eicosanoids in cancer initiation, progression, and metastasis. We have recently developed a novel Fads1 knockout mouse model that allows for the investigation of AA-dependent eicosanoid deficiency without the complication of essential fatty acid deficiency. Interestingly, the survival rate of Fads1-null mice is severely compromised after 2 months on a semi-purified AA-free diet, which precludes long-term chemoprevention studies. Therefore, in this study, dietary AA levels were titrated to determine the minimal level required for survival, while maintaining a distinct AA-deficient phenotype. Null mice supplemented with AA (0.1%, 0.4%, 0.6%, 2.0%, w/w) in the diet exhibited a dose-dependent increase (P < 0.05) in AA, PGE2, 6-keto PGF1α, TXB2, and EdU-positive proliferative cells in the colon. In subsequent experiments, null mice supplemented with 0.6% AA diet were injected with a colon-specific carcinogen (azoxymethane) in order to assess cancer susceptibility. Null mice exhibited significantly (P < 0.05) reduced levels/multiplicity of aberrant crypt foci (ACF) as compared with wild-type sibling littermate control mice. These data indicate that (i) basal/minimal dietary AA supplementation (0.6%) expands the utility of the Fads1-null mouse model for long-term cancer prevention studies and (ii) that AA content in the colonic epithelium modulates colon cancer risk. Cancer Prev Res; 9(9); 750-7. ©2016 AACR.
Assuntos
Ácido Araquidônico/metabolismo , Neoplasias do Colo/fisiopatologia , Modelos Animais de Doenças , Animais , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ácidos Graxos Dessaturases/deficiência , Camundongos , Camundongos KnockoutRESUMO
Epidermal growth factor receptor (EGFR)-mediated signaling is required for optimal intestinal wound healing. Since n-3 polyunsaturated fatty acids (PUFA), specifically docosahexaenoic acid (DHA), alter EGFR signaling and suppress downstream activation of key signaling pathways, we hypothesized that DHA would be detrimental to the process of intestinal wound healing. Using a mouse immortalized colonocyte model, DHA uniquely reduced EGFR ligand-induced receptor activation, whereas DHA and its metabolic precursor eicosapentaenoic acid (EPA) reduced wound-induced EGFR transactivation compared with control (no fatty acid or linoleic acid). Under wounding conditions, the suppression of EGFR activation was associated with a reduction in downstream activation of cytoskeletal remodeling proteins (PLCγ1, Rac1, and Cdc42). Subsequently, DHA and EPA reduced cell migration in response to wounding. Mice were fed a corn oil-, DHA-, or EPA-enriched diet prior to intestinal wounding (2.5% dextran sodium sulfate for 5 days followed by termination after 0, 3, or 6 days of recovery). Mortality was increased in EPA-fed mice and colonic histological injury scores were increased in EPA- and DHA-fed mice compared with corn oil-fed (control) mice. Although kinetics of colonic EGFR activation and downstream signaling (PLCγ1, Rac1, and Cdc42) were delayed by both n-3 PUFA, colonic repair was increased in EPA- relative to DHA-fed mice. These results indicate that, during the early response to intestinal wounding, DHA and EPA uniquely delay the activation of key wound-healing processes in the colon. This effect is mediated, at least in part, via suppression of EGFR-mediated signaling and downstream cytoskeletal remodeling.
Assuntos
Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Receptores ErbB/metabolismo , Processamento de Proteína Pós-Traducional , Cicatrização , Animais , Ácido Araquidônico/metabolismo , Movimento Celular , Células Cultivadas , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Óleo de Milho/administração & dosagem , Sulfato de Dextrana , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/fisiologia , Ácido Eicosapentaenoico/fisiologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Neuropeptídeos/metabolismo , Consumo de Oxigênio , Fosforilação , Transdução de Sinais , Ácido alfa-Linolênico/farmacologia , Ácido alfa-Linolênico/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Arachidonic acid (20:4(Δ5,8,11,14), AA)-derived eicosanoids regulate inflammation and promote cancer development. Previous studies have targeted prostaglandin enzymes in an attempt to modulate AA metabolism. However, due to safety concerns surrounding the use of pharmaceutical agents designed to target Ptgs2 (cyclooxygenase 2) and its downstream targets, it is important to identify new targets upstream of Ptgs2. Therefore, we determined the utility of antagonizing tissue AA levels as a novel approach to suppressing AA-derived eicosanoids. Systemic disruption of the Fads1 (Δ5 desaturase) gene reciprocally altered the levels of dihomo-γ-linolenic acid (20:3(Δ8,11,14), DGLA) and AA in mouse tissues, resulting in a profound increase in 1-series-derived and a concurrent decrease in 2-series-derived prostaglandins. The lack of AA-derived eicosanoids, e.g., PGE2 was associated with perturbed intestinal crypt proliferation, immune cell homeostasis, and a heightened sensitivity to acute inflammatory challenge. In addition, null mice failed to thrive, dying off by 12 weeks of age. Dietary supplementation with AA extended the longevity of null mice to levels comparable to wild-type mice. We propose that this new mouse model will expand our understanding of how AA and its metabolites mediate inflammation and promote malignant transformation, with the eventual goal of identifying new drug targets upstream of Ptgs2.
Assuntos
Modelos Animais de Doenças , Eicosanoides/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Animais , Ácido Araquidônico/administração & dosagem , Dessaturase de Ácido Graxo Delta-5 , Suplementos Nutricionais , Eicosanoides/deficiência , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Dessaturases/genética , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Both fish oil (FO) and curcumin have potential as anti-tumour and anti-inflammatory agents. To further explore their combined effects on dextran sodium sulphate (DSS)-induced colitis, C57BL/6 mice were randomised to four diets (2 × 2 design) differing in fatty acid content with or without curcumin supplementation (FO, FO+2 % curcumin, maize oil (control, MO) or MO+2 % curcumin). Mice were exposed to one or two cycles of DSS in the drinking-water to induce either acute or chronic intestinal inflammation, respectively. FO-fed mice exposed to the single-cycle DSS treatment exhibited the highest mortality (40 %, seventeen of forty-three) compared with MO with the lowest mortality (3 %, one of twenty-nine) (P = 0·0008). Addition of curcumin to MO increased (P = 0·003) mortality to 37 % compared with the control. Consistent with animal survival data, following the one- or two-cycle DSS treatment, both dietary FO and curcumin promoted mucosal injury/ulceration compared with MO. In contrast, compared with other diets, combined FO and curcumin feeding enhanced the resolution of chronic inflammation and suppressed (P < 0·05) a key inflammatory mediator, NF-κB, in the colon mucosa. Mucosal microarray analysis revealed that dietary FO, curcumin and FO plus curcumin combination differentially modulated the expression of genes induced by DSS treatment. These results suggest that dietary lipids and curcumin interact to regulate mucosal homeostasis and the resolution of chronic inflammation in the colon.
Assuntos
Colite/dietoterapia , Colo/metabolismo , Curcumina/uso terapêutico , Citocinas/metabolismo , Suplementos Nutricionais , Óleos de Peixe/uso terapêutico , Regulação da Expressão Gênica , Doença Aguda , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/uso terapêutico , Doença Crônica , Colite/imunologia , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Curcumina/efeitos adversos , Citocinas/genética , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Óleos de Peixe/efeitos adversos , Perfilação da Expressão Gênica , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Irritantes/administração & dosagem , Irritantes/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Distribuição Aleatória , Análise de SobrevidaRESUMO
The biological properties of polyunsaturated fatty acid (PUFA) classes have been the source of much contention. For example, n-3 PUFA are chemoprotective, whereas n-6 PUFA may promote tumor development. Since dietary components can have combinatorial effects, we further examined the apoptotic properties of n-3 or n-6 fatty acids when combined with different fiber sources. Mice were fed diets supplemented with either fish oil (FO; enriched in n-3 PUFA) or corn oil (CO; enriched in n-6 PUFA) and nonfermentable (cellulose) or fermentable (pectin) fiber sources. In complementary experiments, immortalized young adult mouse colonic (YAMC) cells were treated with docosahexaenoic acid (DHA; 22:6n-3) or linoleic acid (LA; 18:2n-6) with or without butyrate. Mice fed a FO and pectin diet had significantly (p < 0.05) increased levels of apoptosis in colonocytes compared to all other diets. Similarly, apoptosis was highly induced in DHA and butyrate cotreated YAMC cells. In contrast, in both YAMC and mouse models, LA/CO with butyrate/pectin treatment reduced apoptosis and enhanced expression of bcl-2. The LA and butyrate induced antiapoptotic phenotype was reversed by knocking down bcl-2 using targeted siRNA. In comparison, overexpression of bcl-2 blocked the proapoptotic effect of DHA and butyrate. These data provide new mechanistic insights into the regulation of apoptosis by dietary PUFA and fiber.
Assuntos
Butiratos/farmacologia , Colo/efeitos dos fármacos , Ácido Linoleico/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Butiratos/administração & dosagem , Células Cultivadas , Celulose/administração & dosagem , Celulose/farmacologia , Colo/citologia , Colo/metabolismo , Óleo de Milho/administração & dosagem , Óleo de Milho/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Sinergismo Farmacológico , Óleos de Peixe/administração & dosagem , Óleos de Peixe/farmacologia , Expressão Gênica/efeitos dos fármacos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Ácido Linoleico/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Pectinas/administração & dosagem , Pectinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND. Besides their health benefits, dietary omega-3 fatty acids (n-3 PUFAs) can impair host resistance to intracellular pathogens. Previously, we and others have showed that n-3 PUFA-treated macrophages poorly control Mycobacterium tuberculosis infection in vitro. METHODS. Wild-type and fat-1 transgenic mice were infected with virulent H37Rv M. tuberculosis via the aerosol route. We evaluated bacteriological and histopathological changes in lungs, as well as differences in activation and antimycobacterial capacity in primary macrophages ex vivo. RESULTS. fat-1 mice were more susceptible to tuberculosis, as demonstrated by higher bacterial loads and less robust inflammatory responses in lungs. Macrophages obtained from fat-1 mice were more readily infected with M. tuberculosis in vitro, compared with wild-type macrophages. This impaired bacterial control in cells from fat-1 mice correlated with reduced proinflammatory cytokine secretion, impaired oxidative metabolism, and diminished M. tuberculosis-lysotracker colocalization within phagosomes. CONCLUSIONS. We showed that endogenous production of n-3 PUFAs in fat-1 mice increases their susceptibility to tuberculosis, which could be explained in part by diminished activation and antimycobacterial responses in cells from fat-1 mice. These data suggest that n-3 PUFA-supplemented diets might have a detrimental effect on immunity to M. tuberculosis and raise concerns regarding the safety of omega-3 dietary supplementation in humans.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Macrófagos/microbiologia , Tuberculose Pulmonar/imunologia , Aminas/metabolismo , Animais , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Nozes , Estresse Oxidativo , Fagossomos , Transporte Proteico , Coloração e Rotulagem , Fatores de Tempo , Tuberculose Pulmonar/microbiologia , Aumento de PesoRESUMO
Phytochemicals may reduce chronic inflammation and cancer risk in part by modulating T-cell nuclear factor-kappaB (NF-kappaB) activation. Therefore, we examined the effects of curcumin (Cur) and limonin (Lim) feeding on NF-kappaB-dependent CD4(+) T-cell proliferation. DO11.10 transgenic mice (n = 5-7) were fed diets containing 1% Cur or 0.02% Lim combined with either (n-6) PUFA [5% corn oil (CO)] or (n-3) PUFA [4% fish oil+1% corn oil (FO)] for 2 wk, followed by splenic CD4(+) T-cell isolation and stimulation with ovalbumin peptide 323-339 (OVA) and antigen-presenting cells from mice fed a conventional nonpurified rodent diet. Both Cur and Lim diets suppressed (P < 0.05) NF-kappaB p65 nuclear translocation in activated CD4(+) T-cells. In contrast, activator protein-1 (c-Jun) and nuclear factor of activated T-cells c1 were not affected compared with the CO control diet (no Cur or Lim). CD4(+) T-cell proliferation in response to either mitogenic anti-CD3/28 monoclonal antibodies (mAb) or antigenic stimulation by OVA was also suppressed (P < 0.05) by Cur as assessed by carboxyfluorescein succinimidyl ester staining. In contrast, interleukin-2 production was not directly associated with NF-kappaB status. Interestingly, dietary combination with FO enhanced the suppressive effects (P < 0.05) of Cur or Lim with respect to CD4(+) T-cell proliferation in response to anti-CD3/28 mAb. These results suggest that combination chemotherapy (FO+Cur or Lim) may favorably modulate CD4(+) T-cell-mediated inflammation.
Assuntos
Linfócitos T CD4-Positivos/citologia , Divisão Celular/efeitos dos fármacos , Curcumina/administração & dosagem , Dieta , Interleucina-2/biossíntese , Limoninas/administração & dosagem , Animais , Anticorpos Monoclonais/farmacologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Óleo de Milho/administração & dosagem , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-6/administração & dosagem , Óleos de Peixe/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , NF-kappa B/fisiologia , Ovalbumina/farmacologia , Fragmentos de Peptídeos/farmacologiaRESUMO
It is well established that the nutritional status of the host affects resistance to disease. The impact of dietary lipids on experimental pulmonary infection with mycobacteria has not been investigated. Therefore, the purpose of this study was to determine the role of dietary (n-3) and (n-6) fatty acids on immunity and resistance to aerosol infection with virulent Mycobacterium tuberculosis in guinea pigs. Weanling guinea pigs were fed purified, isocaloric diets differing only in lipid source, and the effects of diet on specific immune cell functions were evaluated after 3 or 6 wk. Dietary (n-3) fatty acid consumption reduced in vivo skin test and in vitro lympho-proliferative responses (P < 0.05) relative to (n-6) fatty acid consumption. The effect of diet on resistance to mycobacterial infection was assessed by enumerating viable mycobacteria in the lungs and spleens of guinea pigs infected with virulent M. tuberculosis by the aerosol route. (n-3) Fatty acid-fed guinea pigs had more bacteria in the lungs compared with (n-6) fatty acid-fed guinea pigs at 3 (P < 0.05) and 6 wk postinfection (P < 0.01). These data document the immunomodulatory effects of (n-3) fatty acid consumption in the context of tuberculosis resistance. The loss of antigen-specific T-cell functions in addition to impaired resistance to mycobacterial disease suggests a susceptible phenotype in (n-3) fatty acid-fed guinea pigs.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Mycobacterium tuberculosis , Tuberculose/prevenção & controle , Animais , Antígenos de Bactérias/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Dieta , Feminino , Regulação da Expressão Gênica , Cobaias , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Masculino , Mitógenos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The plasma membranes of all eukaryotic cells contain heterogeneous self-organising intrinsically unstable liquid ordered domains or lipid assemblies in which key signal transduction proteins are localised. These assemblies are classified as 'lipid rafts' (10-200 nm), which are composed mostly of cholesterol and sphingolipid microdomains and therefore do not integrate well into the fluid phospholipid bilayers. In addition, caveolae represent a subtype of lipid raft macrodomain that form flask-shaped membrane invaginations containing structural proteins, i.e. caveolins. With respect to the diverse biological effects of long-chain PUFA, increasing evidence suggests that n-3 PUFA and perhaps conjugated fatty acids uniquely alter the basic properties of cell membranes. Because of its polyunsaturation, DHA and possibly conjugated linoleic acid are sterically incompatible with sphingolipid and cholesterol and, therefore, appear to alter lipid raft behaviour and protein function. The present review examines the evidence indicating that dietary sources of n-3 PUFA can profoundly alter the biochemical make up of lipid rafts/caveolae microdomains, thereby influencing cell signalling, protein trafficking and cell cytokinetics.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Membrana Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Humanos , Ácidos Linoleicos Conjugados/farmacologia , Microdomínios da Membrana/efeitos dos fármacosRESUMO
Butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis in part via a Fas-mediated (extrinsic) pathway. In previous studies, we demonstrated that docosahexaenoic acid (DHA, 22:6(Delta4,7,10,13,16,19)) enhances the effect of butyrate by increasing mitochondrial lipid oxidation and mitochondrial Ca(2+)-dependent apoptosis in the colon. In this study, we further examined the mechanism of DHA-butyrate synergism in 1) human colon tumor (HCT-116 isogenic p53+/+ vs. p53-/-) cells and 2) primary cultures of rat colonic crypts. Herein, we show that DHA and butyrate promote apoptosis by enhancing mitochondrial Ca(2+) accumulation in both isogenic cell lines. Ca(2+) accumulation and apoptosis were inhibited by blockade of mitochondrial uniporter-mediated Ca(2+) uptake. In addition, Mito-Q, a mitochondria-targeted antioxidant, also blocked apoptosis induced by DHA and butyrate. In complementary experiments, rats were fed diets supplemented with either corn oil (control, contains no DHA) or fish oil (contains DHA). Colonic crypts were isolated and incubated with or without butyrate, after which the mitochondria-to-cytosol Ca(2+) ratio and crypt viability were measured. No significant difference (P > 0.05) in basal mitochondrial Ca(2+) levels was observed between fish oil- or corn oil-fed animals. In contrast, when fish oil was the dietary lipid source, crypts incubated with butyrate exhibited a significant increase (3.6-fold, P < 0.001) in mitochondrial Ca(2+) compared with corn oil plus butyrate treatment. On the basis of these data, we propose that the combination of DHA and butyrate compared with butyrate alone further enhances colonocyte apoptosis by inducing a p53-independent, oxidation-sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway.
Assuntos
Apoptose/fisiologia , Butiratos/farmacologia , Colo/fisiologia , Ácidos Docosa-Hexaenoicos/farmacologia , Mucosa Intestinal/fisiologia , Mitocôndrias/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colo/citologia , Colo/efeitos dos fármacos , Neoplasias do Colo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Compostos de Rutênio/farmacologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
In recent years, our understanding of the plasma membrane has changed considerably as our knowledge of lipid microdomains has expanded. Lipid microdomains include structures known as lipid rafts and caveolae, which are readily identified by their unique lipid constituents. Cholesterol, sphingolipids and phospholipids with saturated fatty acyl chain moieties are highly enriched in these lipid microdomains. Lipid rafts and caveolae have been shown to play an important role in the compartmentalization, modulation and integration of cell signaling. Therefore, these microdomains may have an influential role in human disease. Dietary n-3 polyunsaturated fatty acids (PUFA) ameliorate a number of human diseases including coronary heart disease, autoimmune and inflammatory disorders, diabetes, obesity and cancer, which has been generally linked to its membrane remodeling properties. Recent in vitro evidence suggests that perturbations in membrane composition alter the function of resident proteins and, consequently, cellular responses. This review examines the role of n-3 PUFA in modulating the lipid composition and functionality of lipid microdomains and its potential significance to human health.
Assuntos
Ácidos Graxos Voláteis/química , Microdomínios da Membrana/química , Animais , Cavéolas/fisiologia , Gorduras Insaturadas na Dieta/farmacologia , Humanos , Modelos BiológicosRESUMO
To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Imunossupressores/farmacologia , Interleucina-2/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Microdomínios da Membrana/imunologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T/imunologia , Animais , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Ácidos Graxos/metabolismo , Feminino , Interleucina-2/biossíntese , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Proteína Quinase C-theta , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/metabolismoRESUMO
Dietary n-3 PUFAs have been shown to attenuate T-cell-mediated inflammation. To investigate whether dietary n-3 PUFAs promote activation-induced cell death (AICD) in CD4+ T-cells induced in vitro to a polarized T-helper1 (Th1) phenotype, C57BL/6 mice were fed diets containing either 5% corn oil (CO; n-6 PUFA control) or 4% fish oil (FO) plus 1% CO (n-3 PUFA) for 2 weeks. Splenic CD4+ T-cells were cultured with alpha-interleukin-4 (alphaIL-4), IL-12, and IL-2 for 2 days and then with recombinant (r) IL-12 and rIL-2 for 3 days in the presence of diet-matched homologous mouse serum (HMS) to prevent loss of cell membrane fatty acids, or with fetal bovine serum. After polarization, Th1 cells were reactivated and analyzed for interferon-gamma and IL-4 by intracellular cytokine staining and for apoptosis by Annexin V/propidium iodide. Dietary FO enhanced Th1 polarization by 49% (P = 0.0001) and AICD by 24% (P = 0.0001) only in cells cultured in the presence of HMS. FO enhancement of Th1 polarization and AICD after culture was associated with the maintenance of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) in plasma membrane lipid rafts. In conclusion, n-3 PUFAs enhance the polarization and deletion of proinflammatory Th1 cells, possibly as a result of alterations in membrane microdomain fatty acid composition.
Assuntos
Gorduras Insaturadas na Dieta/metabolismo , Ácidos Graxos Insaturados/metabolismo , Células Th1/metabolismo , Triglicerídeos/metabolismo , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Ácidos Graxos Ômega-3 , Microdomínios da Membrana/metabolismo , Camundongos , Células Th1/citologiaRESUMO
The underlying mechanisms by which n-3 polyunsaturated fatty acids (PUFA) exert a chemopreventive effect in the colon have not been elucidated. Retinoid X receptors (RXR) are a family of nuclear receptors implicated in cancer chemoprevention. Since docosahexaenoic acid (DHA), an n-3 PUFA enriched in fish oil, reduces colonocyte proliferation and enhances apoptosis relative to n-6 PUFA-treated cells, we determined whether DHA can serve as a specific ligand for RXRalpha activation relative to n-6 PUFA in colonocytes. In a mammalian one-hybrid assay, immortalized young adult mouse colonic (YAMC) cells were co-transfected with a yeast galactose upstream activating sequence (UAS)4-tk-Luciferase (Luc) reporter plasmid, plus either GAL4 DNA-binding domain fused to RXRalpha, retinoic acid receptor alpha or GAL4 alone, followed by an n-3, n-6 or n-9 fatty acid incubation. Luc activity levels were dose-dependently elevated only in n-3 PUFA (DHA)-treated RXRalpha. Since RXR homodimers and RXR/peroxisome proliferator-activated receptor (PPAR) heterodimers bind consensus direct repeat (DR1) motifs, YAMC and NCM460 (a normal human colonic cell line), were respectively, co-transfected with RXRalpha and DR1-Luc, followed by different PUFA treatment. Luc activity levels were increased (P < 0.05) only in DHA groups. The DHA-dependent induction of DR-1-Luc was reduced to basal levels upon RXRalpha antagonist-treatment, with no effect on PPARgamma antagonist-treatment. A role for select RXR isoforms in colonocyte biology was also determined by examining nuclear receptor mRNA levels in rat colon following dietary lipid and carcinogen exposure over time. RXRalpha, RXRbeta and RXRgamma were detected in rat colonic mucosa, and the levels of RXRalpha and RXRgamma were elevated in fish oil (n-3 PUFA) versus corn oil (n-6 PUFA) fed animals after 16 weeks. These data indicate that, RXRalpha, an obligatory component of various nuclear receptors, preferentially binds n-3 PUFA in colonocytes, and that the nuclear receptor targets for PUFA in the colon are modulated by dietary lipid exposure.
Assuntos
Colo/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Receptores do Ácido Retinoico/metabolismo , Animais , Linhagem Celular , Gorduras na Dieta , Humanos , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptor alfa de Ácido Retinoico , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
We have shown that a combination of fish oil (high in n-3 fatty acids) with the butyrate-producing fiber pectin, upregulates apoptosis in colon cells exposed to the carcinogen azoxymethane, protecting against colon tumor development. We now hypothesize that n-3 fatty acids prime the colonocytes such that butyrate can initiate apoptosis. To test this, 30 Sprague-Dawley rats were provided with diets differing in the fatty acid composition (corn oil, fish oil or a purified fatty acid ethyl ester diet). Intact colon crypts were exposed ex vivo to butyrate, and analyzed for reactive oxygen species (ROS), mitochondrial membrane potential (MMP), translocation of cytochrome C to the cytosol, and caspase-3 activity (early events in apoptosis). The fatty acid composition of the three major mitochondrial phospholipids was also determined, and an unsaturation index calculated. The unsaturation index in cardiolipin was correlated with ROS levels (R = 0.99; P = 0.02). When colon crypts from fish oil and FAEE-fed rats were exposed to butyrate, MMP decreased (P = 0.041); and translocation of cytochrome C to the cytosol (P = 0.037) and caspase-3 activation increased (P = 0.032). The data suggest that fish oil may prime the colonocytes for butyrate-induced apoptosis by enhancing the unsaturation of mitochondrial phospholipids, especially cardiolipin, resulting in an increase in ROS and initiating apoptotic cascade.